Immunosuppressant compounds, methods and uses related thereto

ABSTRACT

The present invention relates to compositions and methods for suppressing an immune response, e.g., by inhibiting class II MHC-mediated activation of T cells. The subject compounds and methods may be used to treat or prevent disorders such as rheumatoid arthritis and/or multiple sclerosis.

BACKGROUND OF THE INVENTION

MHC molecules exist in two forms, class I and class II, both encodedwithin a single gene complex. MHC genes are highly polymorphic: mostloci have up to about 100 alleles in the human population (Hansen, T.H., et al. 1993 In “Fundamental Immunology” Ed. Paul, W. E., RavenPress,New York, N.Y., p. 577).

Class I MHC molecules are 45 kD transmembrane glycoproteins,noncovalently associated with another glycoprotein, the 12 kD beta-2microglobulin. The latter is not inserted into the cell membrane, and isencoded outside the MHC. Human class I molecules are of three differentisotypes, termed HLA-A, -B, and -C, encoded in separate loci. The tissueexpression of class I molecules is ubiquitous and codominant. Thethree-dimensional structure of several human and murine class Imolecules have been resolved (Bjorkman, P. J., et al. (1987) Nature,329, 506; Garrett, T. P. J., et al. (1989) Nature, 342, 692; Madden, D.R., et al. (1991) Nature, 353, 321; Fremont, D. H., et al. (1992)Science, 257, 919). Their first and second extracellular domains foldinto a binding site consisting of a β-pleated sheet floor flanked by twoparallel α-helical portions. The binding site presents 7-9 amino acidlong antigenic peptides to cytotoxic effector T lymphocytes (Tc) (Maddenet al. and Fremont et al., above). Most of these peptides arise fromproteins synthesized inside the antigen presenting cell (APC), e.g.,from proteins of viruses or other intracellular parasites, or frommisfolded self proteins. The three class I isotypes, as well as theirallelic forms, have different peptide binding specificities, dependingon polymorphic residues within the binding site (Falk, K, et al. (1991)Nature, 351, 290; Falk, K, et al. (1992) Eur. J. Immunol., 22,277).There is an additional binding site on the third class I domain thatinteracts with CD8 molecules expressed selectively on Tc cells. Theinitial step in Tc cell activation is the simultaneous interaction oftheir antigen receptor (TCR) with the presented peptide and CD8 with itsacceptor site on the same class I molecule.

Class II MHC molecules are noncovalently associated heterodimers of twotransmembrane glycoproteins, the 35 kD α chain and the 28 kD β chain. Inhumans, class II molecules occur as three different isotypes, termedHLA-DP, -DQ, and -DR. Polymorphism in DR is restricted to the β chain,whereas both chains are polymorphic in the DP and DQ isotypes. Class IImolecules are expressed codominantly, but in contrast to class I,exhibit a restricted tissue distribution: they are present only on thesurface of cells of the immune system (constitutive expression on Blymphocytes and dendritic cells, and inducible expression on T cells andmonocytes). The three-dimensional structure of three different DRmolecules has been determined (Brown, J. H., et al. (1993), Nature, 364,33; Stern, L. J., et al. (1994) Nature, 388, 215; Ghosh, P., et al.(1995) Nature, 378, 457; Dessen, A., et al. (1997) Immunity, 7, 473).Overall, their structure is very similar to that of class I molecules.The peptide binding site is composed of the first domains of α nd βchain, which, in contrast to class I, is open on both sides, allowingthe binding of longer (12-24 residues long) peptides (Chicz, R. M., etal. (1992) Nature, 358, 764). An additional binding site on the seconddomain of P chains interacts with the CD4 molecule, expressedselectively on helper T (Th) cells. This molecule has a co-receptorfunction for Th cells, analogous to that of CD8 for Tc cells. Duringtheir biosynthesis and intracellular transport, class II heterodimersare chaperoned by a third, nonpolymorphic non-MHC-encoded 31 kD protein,termed invariant (Ii) chain (Cresswell, P. (1994) Annu. Rev. Immunol.,12, 259). The Ii chain shields the peptide binding site of class IImolecules during their transport in the cytosol, until they reach anacidic endosomal compartment, where it is cleaved by proteases, leavingonly a peptide thereof, termed CLIP, in the binding site. The exchangeof CLIP with antigenic peptides is catalysed by another MHC-encodedmolecule, termed HLA-DM, in the endosome (Vogt, A. B., et al. (1996)Proc. Natl. Acad. Sci. USA. 93, 9724). The antigenic peptides derivemostly from endocytosed external proteins (Germain, R. N. (1994) Cell,76, 287).

The nature of interaction between DR molecules and peptides is largelyunderstood. There is one major pocket in the binding site that iscritical for the interaction with a hydrophobic anchor residue of thepeptide, and additional minor pockets containing polymorphic 0 chainresidues, which confer a degree of allotype-specificity to peptidebinding (Stern et al., above; Hammer, J., et al. (1993) J. Exp. Med.,176, 1007; Hammer, J., et al. (1994) Cell. 74,197; Hammer, J., et al.(1994) Proc. Natl. Acad. Sci., USA 91, 4456; Hammer, J., et al. (1995)J. Exp. Med., 180, 2353). The peptide main chain also forms importanthydrogen bonds with side chains of certain conserved residues in thebinding site, which determine the overall conformation and side chainorientation of the bound peptide (Stern et al., above).

A large body of evidence has demonstrated that susceptibility to manydiseases, in particular autoimmune diseases, is strongly associated withspecific alleles of the major histocompatibility complex (reviewed inTiwari, J., and Terasaki, P. (1985), HLA and disease association (NewYork; Springer Verlag)). Although some class I-associated diseasesexist, most autoimmune conditions have been found to be associated withclass II alleles. For example, class II alleles DRB1*0101, 0401, 0404,and 0405 occur at increased frequency among rheumatoid arthritis (RA)patients (McMichael, S. J., et al. (1977) Arthritis Rheum., 20, 1037;Stasny, P. (1978) N. Engl. J. Med., 298, 869; Ohta, N., et al. (1982)Hum. Immunol., 5, 123; Schiff, B., et al. (1982) Ann. Rheum. Dis., 41,403), whereas DRB1*1501 is associated with multiple sclerosis (MS), andthe DQ allele combination DQA1*0301/B1*0302 with insulin-dependentdiabetes mellitus (IDDM). In RA, altogether >94% of rheumatoid factorpositive patients carry one of the susceptibility alleles (Nepom, G. T.,et al. (1989) Arthritis, Rheum., 32, 15).

The effect of DRB1 alleles on RA is manifested in different ways: first,the disease association shows ethnic-dependent preference for one or theother allele (Ohta et al., and Schiff et al., above), second, DRB1*0401is associated with more severe forms of the disease than the otheralleles (Lanchbury, J. S., et al. (1991) Hum. Immunol., 32, 56), andthird, a gene dosage affect can be observed, in that homozygosity for asusceptibility allele or combinations of two susceptibility allelesconfer more severe, chronic forms or juvenile onset of RA (Wordworth,P., et al. (1992) Am. J. Hum. Genet., 51, 585; Nepom, B. S. (1993) Clin.Immunol. Immunopathol., 67, 850). The latter finding indicates that theDRB1 locus can control both initiation and progression of the disease.

The DRB chains encoded by RA-linked DRB1 alleles exhibit polymorphicdifferences, but all share a stretch of identical, or almost identicalamino acid sequence at positions 67-74, known as the “shared epitope”(Nepom et al., (1989) above; Gregersen, P. K., et al. (1987) ArthritisRheum. 30, 1205). Residues in the shared epitope region contribute tothe formation of the α helix on one side of the peptide binding groove(Brown et al., Stern et al., and Dessen et al., above), and are thusexpected to influence peptide binding. Indeed, the basic residue Lys orArg at position (p)71 of RA-associated DR allotypes imparts selectivityon peptide binding by favoring negative and disfavoring positive chargeat residue p4 of the displayed peptide, whereas the RA-unlinked allotypeDRB1*0402 with acidic residues Asp and Glu at p70 and 71 shows theopposite charge preference at residue p4 of the displayed peptide(Hammer, J., et al. (1995) J. Exp. Med., 181, 1847). Although theautoantigens inducing RA remain unknown, several joint cartilageproteins have peptide sequences which can selectively bind toRA-associated DR molecules due to an acidic residue at p4 (Dessen etal., Hammer et al., (1995) above). These proteins can thus be candidateantigens for an autoimmune response causing RA pathology (Rosloniec, E.F., et al. (1997) J. Exp. Med. 185, 1113). The opposite (positive)charge preference of DRB1-0402 has been shown to confer selectivepresentation of peptides with a basic residue at p4, derived fromdesmoglein 3, an autoantigen involved in the 0402-associated autoimmunedisease, pemphigus vulgaris (Wucherpfennig, K. W., et al. (1995) Proc.Natl. Acad, Sci. USA, 92, 11935). These data strongly support thehypothesis that selective presentation of autoantigenic peptides bydisease-linked MHC allotypes could be the mechanism underlying thegenetic association between DRB1 alleles and autoimmune diseases (Todd,J. A., et al. (1988) Science, 240, 1003). The disease process itself isdriven by Th cells recognizing such peptides. The activated autoreactiveTh cells secrete different pro-inflammatory cytokines, which in turnattract further inflammatory cells to the site, and cause a chronicinflammation in the affected organ.

Of the two classes of MHC molecules, class II is the primary target forimmunosuppressive intervention for the following reasons: First, MHC-IImolecules activate T helper (Th) cells that are central toimmunoregulation, and are responsible for most of the immunopathology ininflammatory diseases. Second, most autoimmune diseases are geneticallyassociated with class II alleles. Third, under normal physicological ornon-pathological conditions, MC-II molecules are expressed selectivelyon cells of the immune system, whereas MHC-I are present on most somaticcells.

Peptide binding to class II (e.g., DR) molecules requires the presenceof defined side chains at so-called “anchor positions” of the displayedpeptide, which all together form a particular binding motif; however, atnon-anchor positions, a variation of side chains is permitted withoutinfluence on binding (Hammer et al., (1993, 1994, and 1995), above).This binding mechanism enables the presentation of many differentpeptides by a given allotype. The side chains at anchor positionsinteract with specific pockets within the binding site, whereas those atnon-anchor positions point outward, and are available for recognition bythe TCR of Th cells. It is therefore conceivable that replacement ofautoantigenic peptides presented by autoimmune disease-associated MHCmolecules by a compound having the same binding motif but beingdifferent at non-anchor positions could prevent the activation ofautoimmune T cells, and thus interrupt the disease process. Themechanism whereby such a compound would exert its effect is competitiveantagonism for the antigen-presenting site. Compounds bindingselectively to class II molecules involved in a particular autoimmunedisease are therefore expected to interfere specifically with thatdisease. Additional peptides which bind to MHC molecules and inhibit Tcell activation have been disclosed in, for example, InternationalPatent Applications WO 92/02543, WO 93/05011, and WO 95/07707.

A pharmaceutical agent targeting class II MHC molecules would offerseveral advantages over most available immunosuppressive drugs. First,it would represent a disease mechanism-based intervention, which isexpected to interrupt the initial event in the pathogenic cascade.Second, it can be designed to be selective for only a few class IIallotypes, i.e., binding with improved affinity to those allotypesassociated with disease, leaving the remainder of the antigen presentingsystem available for protective responses against pathogens, andtherefore causing fewer immunocompromising side effects than mostimmunosuppressive drugs. Third, the methods and compounds could beapplied without any specific knowledge of the actual autoantigenscausing the disease. Finally, it would be advantageous if such apharmaceutical agent showed superior stability in certain biologicalenvironments. For example, high drug stability in mammalian plasmas suchas rat, mouse or human plasma, would be desirable given that many cellsof the immune system are found in the blood together with powerfulpeptide degrading enzymes. High drug stability in rodent plasma,especially rat plasma, is particularly advantageous since mosttherapeutics are initially tested for efficacy, toxicity, and/orpharmacokinetics in rodent models or systems. Drug stability againstCathepsin degradation is equally desirable since mechanism-basedtherapeutic intervention requires that pharmaceutical agents targetingclass II MHC molecules may be endocytosed and transported within thecell using Cathepsin-containing endosomes before presentation to the MHCII molecule.

SUMMARY OF THE INVENTION

The present invention relates to compounds, pharmaceutical compositionsand methods for suppressing an immune response, e.g., by inhibitingclass II MHC-mediated activation of T cells. The compounds disclosedbelow, which include an unnatural arginine substitute in the compoundsof Formula II, may exhibit increased stability in blood plasma (e.g.,mouse and rat plasma) and increased binding affinity to MHC-class IImolecules of interest (0401, 0101 and 0404) by as much as a factor of1.25-3, as compared to corresponding compounds containing Arg in theposition of the substitute amino acid. Further compounds disclosed belowcomprise a terminating group in the compounds of Formula I. Suchcompounds of Formulae I or II may also show increased in vivo inhibitionof T-cell response by as much as a factor of 1.25-3. Such compounds mayshow effective immunosuppression in mouse models of certain immunedisorders. The subject compounds and methods may be used to treatdisorders such as rheumatoid aritis and/or multiple sclerosis.

In certain embodiments, the subject compounds are used for thepreparation of a pharmaceutical composition for the treatment of ananimal, such as a human, e.g., to treat or prevent a conditioncharacterized by MHC class II-mediated activation of T cells, or byexpression of MHC class II protein at a pathological site ofinflammation, such as an autoimmune disease. The subject compoundsand/or compositions may be used in the treatment or prevention of suchdiseases, including those enumerated specifically below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Improved binding of a Gpg (guanylpiperidyl glycine)-containingheptamer compound of the invention (P53) to MHC class II protein 0401compared to the Arg-containing equivalent (P51). A published leadpeptide (P3; Falcioni et al 1999; Nature Biotech 17, 562-567) is used asa positive control. Using standard statistical software, non-linearlogistic regression curves were fitted to replica data points generatedaccording to Example 14. IC50s were estimated from the fitted curves andare represented by vertical lines of the appropriate line-type (P53solid line, P51 dashed line, P3 dotted line) for the correspondingcompound.

FIG. 2 Improved binding of a Gpg-containing tetramer compound of theinvention (P74) to MHC class II protein 0401 compared to theArg-containing equivalent (P71). A published lead peptide (P3; Falcioniet al 1999) is used as a positive control. Using standard statisticalsoftware, non-linear logistic regression curves were fitted to replicadata points generated according to Example 14. IC50s were estimated fromthe fitted curves and are represented by vertical lines of theappropriate line-type (P74 solid line, P71 dashed line, P3 dotted line)for the corresponding compound.

FIG. 3 Improved binding of a preferred Gpg (guanylpiperidylglycine)-containing tetramer compound of the invention (P69) to MHCclass II protein 0101 compared to the Arg-containing equivalent (P82).Using standard statistical software, non-linear logistic regressioncurves were fitted to replica data points generated according to Example14. IC50s were estimated from the fitted curves and are represented byvertical lines of the appropriate line-type (P69 solid line, P82 dashedline) for the corresponding compound.

FIG. 4 Improved binding of a preferred Gpg (guanylpiperidylglycine)-containing tetramer compound of the invention (P74) to MHCclass II protein 0401 compared to the Arg-containing equivalent (P71).Using standard statistical software, non-linear logistic regressioncurves were fitted to replica data points generated according to Example14. IC50s were estimated from the fitted curves and are represented byvertical lines of the appropriate line-type (P74 solid line, P71 dashedline) for the corresponding compound.

FIG. 5 Improved binding of a preferred Gpg (guanylpiperidylglycine)-containing tetramer compound of the invention (P101) to MHCclass II protein 0401 compared to the Arg-containing equivalent (P98).Using standard statistical software, non-linear logistic regressioncurves were fitted to replica data points generated according to Example14. IC₅₀s were estimated from the fitted curves and are represented byvertical lines of the appropriate line-type (P101 solid line, P98 dashedline) for the corresponding compound.

FIG. 6 Improved binding of a Gpg-containing heptamer compound of theinvention (P47) to MHC class II protein expressed on the surface of LG2cells compared to the Arg-containing equivalent (P43). Using standardstatistical software, non-linear logistic regression curves were fittedto replica data points generated according to Example 15. IC₅₀s wereestimated from the fitted curves and are represented by vertical linesof the appropriate line-type (P47 solid line, P43 dashed line) for thecorresponding compound.

FIG. 7 Improved binding of a Gpg-containing tetramer compound of theinvention CP74) to MHC class II protein expressed on the surface ofPriess cells compared to the Arg-containing equivalent (P71). Usingstandard statistical software, non-linear logistic regression curveswere fitted to replica data points generated according to Example 15.IC₅₀s were estimated from the fitted curves and are represented byvertical lines of the appropriate line-type (P74 solid line, P71 dashedline) for the corresponding compound

FIG. 8 Improved stability (arbitrary units) of Gpg-containing heptamerand tetramer compounds of the invention in rat plasma after 24 hourscompared to the A rg-containing equivalent. Data for the corresponding Gpg/Arg compounds are shown as adjacent bars.

FIG. 9 A dose-respose curve demonstrating improved immunosuppressiveproperties as measured by a T-cell activation assay of P53(Gpg-containing) a preferred heptamer compound of the invention,compared to the Arg-containing peptide (P51). Using standard statisticalsoftware, non-linear logistic regression curves were fitted to replicadata points generated according to Example 19. IC₅₀s were estimated fromthe fitted curves and are represented by vertical lines of theappropriate line-type (P53 solid line, P51 dashed line) for thecorresponding compound.

FIG. 10 Dose-respose curves demonstrating immunosuppressive propertiesas measured by a T-cell activation assay of preferred compounds of theinvention (a) P69, (b) P101, (c) P74 and (d) P53.

FIG. 11 A dose-response curve demonstrating the improvedimmunosuppressive properties as measured by IL-2 secretion of P41-1(Gpg-containing) (squares and dashed line), a heptamer compound of theinvention, compared to the Arg-containing peptide (P40-1) (diamonds andsolid line).

FIG. 12 A dose-response curve demonstrating the improvedimmunosuppressive properties as measured by IL-2 secretion of P69(Gpg-containing, a preferred tetramer compound of the invention,compared to the Arg-containing peptide (P82). Using standard statisticalsoftware, non-linear logistic regression curves were fitted to replicadata points generated according to Example 20. IC₅₀s were estimated fromthe fitted curves and are represented by vertical lines of theappropriate line-type (P69 solid line, P82 dashed line) for thecorresponding compound

FIG. 13 The immunosuppressive properties of P53 (Gpg-containing) asmeasured by IL-2 secretion (squares and solid line), a preferredheptamer compound of the invention, compared to a DMSO control (diamondsand dotted line).

FIG. 14 Superior in-vivo immunosuppressive properties of P69(Gpg-containing) a preferred tetramer compound of the inventionfollowing Co-imunisation with antigen as measured by T-cellproliferation, compared to the Arg-containing equivilent (P82).

FIG. 15 Di-vivo immunosuppressive properties of preferred tetramer andheptamer compounds of the invention following co-imunisation withantigen as measured by T-cell proliferation.

FIG. 16 Efficacy of preferred tetramer and heptamer compounds of theinvention (P69, P53 and P74) in the CIA mouse model for rheumatoidarthritis compared to solvent as control.

FIG. 17 Efficacy of preferred tetramer and heptamer compounds of theinvention (P69, P53 and P74) in the EAE mouse model for multiplesclerosis prevention compared to solvent as control.

FIG. 18 Efficacy of preferred tetramer and heptamer compounds of theinvention (P69 and P53) in the EAE mouse model for multiple sclerosistreatment compared to solvent as control.

FIG. 19 Superior efficacy of a preferred Gpc-containing compound of theinvention (P69) compared to the equivilent Arg containing compound (P82)in the EAE mouse model of multiple sclerosis.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The present invention relates to compounds, such as peptidomimeticcompounds, which can be used to suppress an undesired immune activity,e.g., by inhibiting class II MHC-mediated T cell activation, such as inthe treatment or prevention of autoimmune disorders. In certainembodiments, these compounds are characterized by binding to class IImolecules, their ability to prevent the binding of self antigens or todisplace self antigens already bound to class II molecules and/or theirability to inhibit T cell activation by modulating a class II MHCrestricted immune response by an alternate mode of action. In suchembodiments, compounds of the invention may be termed “inhibitors”,“inhibiting agents”, “subject inhibitors”, “peptiodomimetics” (including“heptamer” and “tetramer” compounds), “compounds of the invention” or“inhibitors of the invention”. In certain embodiments, the preferredclass II molecules are DR isotypes. In preferred embodiments, such aninhibitor is a small molecule, e.g., a compound having a molecularweight less than 2000 amu, preferably less than 1000 amu, even morepreferably less than 700 amu.

The compounds of the invention which inhibit class II MHC activity havetherapeutic value in the prevention or treatment of various class IIMHC-related diseases or disorders. The compounds of the invention may beadministered to a patient for treatment of an immune disorder, forexample, involving undesirable or inappropriate immune activity, or maybe used to prepare a therapeutic medicament. In particular, an effectivedose of an inhibitor of the invention may be therapeutically applied toameliorate or to prevent insulin-dependent diabetes, multiple sclerosis,rheumatoid arthritis, etc. An effective dose of a compound of theinvention for the treatment of a disorder involving undesirable orinappropriate MHC activity, such as an autoimmune disorder, can bedetermined by standard means known in the art taking into accountroutine safety studies, toxicity studies, dose concentration studies andmethod of delivery, e.g., bolus, continuous or repeated. In a particularembodiment, a dose of about 0.01 to about 500 mg/kg can be administered.

II. Definitions

As u sed herein, the term “MHC activity” refers to the ability of an MHCmolecule to stimulate an immune response, e.g., by activating T cells.An inhibitor of MHC activity is capable of suppressing this activity,and thus inhibits the activation of T cells by MHC. In preferredembodiments, a subject inhibitor selectively inhibits activation by aparticular class II MHC isotype or allotype. Such inhibitors may becapable of suppressing a particular undesirable MHC activity withoutinterfering with all MHC activity in an organism, thereby selectivelytreating an unwanted immune response in an animal, such as a mammal,preferably a human, without compromising the animal's immune response ingeneral. Such unwanted immune response may be one associated with aparticular disease such as rheumatoid arthritis or multiple sclerosis.

The term “prodrug” is intended to encompass compounds that, underphysiological conditions, are converted into the inhibitor agents of thepresent invention. A common method for making a prodrug is to selectmoieties which are hydrolyzed under physiological conditions to providethe desired biologically active drug. In other embodiments, the prodrugis converted by an enzymatic activity of the patient or alternatively ofa target pathogen. “Treat”, as used herein, means at least lessening theseverity or ameliorating the effects of, for example, one or moresymptoms, of a disorder or condition.

“Hydrophobic”, as used herein when pertaining to a molecular species,means that in a partitioning experiment, the majority of the moleculesof the molecular species under investigation is retained in the organicrather than the aqueous layer. Preferably, more than about 55%, 75%,85%, or over about 95% of the molecule is retained in the organic layer.Suitable organic solvents for such a partitioning experiment will beknown to a skilled artisan but include, without limitation, octanol,diethylether, dichloromethane, and chloroform. When pertaining to afunctional group or residue, hydrophobic refers to the property of saidfunctional group or residue to increase the hydrophobicity of amolecular species when added to it structurally.

“Prevent”, as used herein, means to delay or preclude the onset of, forexample, one or more symptoms of a disorder or condition.

The term “IC₅₀” means the concentration of a drug which inhibits anactivity or property by 50%, e.g., by reducing the frequency of acondition, such as cell death, by 50%, by reducing binding of acompetitor peptide to MHC II protein by 50% or by reducing the level ofan activity, such as T-cell proliferation or IL2 secretion, by 50%.

The term “ED₅₀” means the dose of a drug that produces 50% of themaximum of a given response or effect. Alternatively, it may refer tothe dose that produces a pre-determined response in 50% of test subjectsor preparations.

The term “LD₅₀” means the dose of a drug that is lethal in 50% of testsubjects.

The term “therapeutic index” refers to the therapeutic index of a drugdefined as LD₅₀/ED₅₀.

The term “patient” refers to an animal, preferably a mammal, includinghumans as well as livestock and other veterinary subjects.

The term “structure-activity relationship” or “SAR” refers to the way inwhich altering the molecular structure of drugs alters their interactionwith a receptor, enzyme, etc.

“Small molecule” refers to a molecule which has a molecular weight ofless than about 2000 amu, or less than about 1000 amu, and even lessthan about 700 amu.

The term “aliphatic” refers to a linear, branched, or cyclic alkane,alkene, or alkyne. In certain embodiments, aliphatic groups in thepresent invention are linear or branched and have from 1 to about 20carbon atoms.

The term “alkyl” refers to the radical of a saturated aliphatic group,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. In certain embodiments, a straightchain or branched chain alkyl has about 30 or fewer carbon atoms in itsbackbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ for branched chain),and alternatively, a bout 20 or fewer. Likewise, cycloalkyls have fromabout 3 to about 10 carbon atoms in their ring structure, andalternatively about 5, 6 or 7 carbons in the ring structure.

Moreover, the term “alkyl” (or “lower alkyl”) includes both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. Such substituents mayinclude, for example, a halogen, a hydroxyl, a carbonyl (such as acarboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (suchas a thioester, a thioacetate, or a thioformate), an alkoxyl, aphosphoryl, a phosphonate, a phosphinate, an amino, an amido, anamidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, analkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, asulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromaticmoiety. It will be understood by those skilled in the art that themoieties substituted on the hydrocarbon chain may themselves besubstituted, if appropriate. For instance, the substituents of asubstituted alkyl may include substituted and unsubstituted forms ofamino, azido, imino, amido, phosphoryl (including phosphonate andphosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl andsulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls(including ketones, aldehydes, carboxylates, and esters), —CF₃, —CN andthe like. Exemplary substituted alkyls are described below. Cycloalkylsmay be further substituted with alkyls, alkenyls, alkoxys, alkylthios,aminoalkyls, carbonyl-substituted alkyls, —CF₃, —CN, and the like.

‘Ci alkyl’ is an alkyl chain having i member atoms. For example, C4alkyls contain four carbon member atoms. C4 alkyls containing may besaturated or unsaturated with one or two double bonds (cis or trans) orone triple bond. Preferred C4 alkyls are saturated. Preferredunsaturated C4 alkyl have one double bond. C4 alkyl may be unsubstitutedor substituted with one or two substituents. Preferred substituentsinclude lower alkyl, lower heteroalkyl, cyano, halo, and haloalkyl.

‘Heteroalkyl’ is a saturated or unsaturated chain of carbon atoms and atleast one heteroatom, wherein no two heteroatoms are adjacent.Heteroalkyl chains contain from 1 to 18 member atoms (carbon andheteroatoms) in the chain, preferably 1 to 12, more preferably 1 to 6,more preferably still 1 to 4. Heteroalkyl chains may be straight orbranched. Preferred branched heteroalkyl have one or two branches,preferably one branch. Preferred heteroalkyl are saturated. Unsaturatedheteroalkyl have one or more double bonds and/or one or more triplebonds. Preferred unsaturated heteroalkyl have one or two double bonds orone triple bond, more preferably one double bond. Heteroalkyl chains maybe unsubstituted or substituted with from 1 to about 4 substituentsunless otherwise specified. Preferred heteroalkyl are unsubstituted.Preferred heteroalkyl substituents include halo, aryl (e.g., phenyl,tolyl, alkoxyphenyl, alkoxycarbonylphenyl, halophenyl), heterocyclyl,heteroaryl. For example, alkyl chains substituted with the followingsubstituents are heteroalkyl: alkoxy (e.g., methoxy, ethoxy, propoxy,butoxy, pentoxy), aryloxy (e.g., phenoxy, chlorophenoxy, tolyloxy,methoxyphenoxy, benzyloxy, alkoxycarbonylphenoxy, acyloxyphenoxy),acyloxy (e.g., propionyloxy, benzoyloxy, acetoxy), carbamoyloxy,carboxy, mercapto, alkylthio, acylthio, arylthio (e.g., phenylthio,chlorophenylthio, alkylphenylthio, alkoxyphenylthio, benzylthio,alkoxycarbonylphenylthio), amino (e.g., amino, mono- and di-C1-C3alkylamino, methylphenylamino, methylbenzylamino, C1-C3 alkylamido,carbamamido, ureido, guanidino).

‘Mi heteroalkyl’ is a heteroalkyl chain having i member atoms. Forexample, M4 heteroalkyls contain one or two non-adjacent heteroatommember atoms. M4 heteroalkyls containing 1 heteroatom member atom may besaturated or unsaturated with one double bond (cis or trans) or onetriple bond. Preferred M 4 heteroalkyl containing 2 heteroatom memberatoms are saturated. Preferred unsaturated M4 heteroalkyl have onedouble bond. M4 heteroalkyl may be unsubstituted or substituted with oneor two substituents. Preferred substituents include lower alkyl, lowerheteroalkyl, cyano, halo, and haloalkyl.

The term “aralkyl” refers to an alkyl group substituted with an arylgroup (e.g., an aromatic or heteroaromatic group).

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

Unless the number of carbons is otherwise specified, “lower alkyl”refers to an alkyl group, as defined above, but having from one to tencarbons, alternatively from one to about six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths.

The term “heteroatom” refers to an atom of any element other than carbonor hydrogen. Illustrative heteroatoms include boron, nitrogen, oxygen,phosphorus, sulfur and selenium, and alternatively oxygen, nitrogen orsulfur.

The term “amino acid” refers to an organic compound bearing both acarboxylic acid group and an amino group, preferably attached to thesame carbon atom or to adjacent carbon atoms, most preferably to thesame carbon atom. Exemplary amino acids are those found in nature, suchas amino acids that are used to synthesize proteins in cells, althoughunnatural amino acids such as those used in the Exemplification orotherwise known in the art are also contemplated. An “amino acidresidue” refers to a derivative of an amino acid wherein either or bothof the amino and carboxylic acid groups have been joined to anothermoiety, e.g., to form an amide, thioamide, sulfonamide, etc.

The term “amino acid analog” includes amino acid-like molecules, orresidues thereof, wherein the carbonyl of the carboxylic acid group isreplaced with another electrophilic moiety, such as a thiocarbonyl orsulfonyl group. The term also includes analogs of dipeptides, such asthe [SΨ(oxaz)L] and [SΨ(imid)L] moieties discussed below, as well asanalogs of dipeptides wherein the internal amide bond is replaced by analkene. Other amino acid analogs suitable for use in the presentinvention are well known to those of skill in the art. Compounds, suchas inhibitors of the invention, that comprise one or more amino acidanalogs are often termed “peptidomimetic” or “mimetic” compounds.

The term “aryl” includes 5-, 6- and 7-membered single-ring aromaticgroups that may include from zero to four heteroatoms, for example,benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole,triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, andthe like. Those aryl groups having heteroatoms in the ring structure mayalso be referred to as “aryl heterocycles” or “heteroaromatics.” Thearomatic ring may be substituted at one or more ring positions with suchsubstituents as described above, for example, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester,heterocyclyl, aromatic or heteroaromatic moieties, —CF₃, —CN, or thelike. The term “aryl” also includes polycyclic ring systems having twoor more cyclic rings in which two or more carbons are common to twoadjoining rings (the rings are “fused rings”) wherein at least one ofthe rings is aromatic, e.g., the other cyclic rings may be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

The terms ortho, meta and Mara apply to 1,2-, 1,3- and 1,4-disubstitutedbenzenes, respectively. For example, the names 1,2-dimethylbenzene andortho-dimethylbenzene are synonymous.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to about10-membered ring structures, alternatively 3- to about 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles mayalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringmay be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle may be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromaticmoiety, —CF₃, —CN, or the like.

The term “carbocycle” refers to an aromatic or non-aromatic ring inwhich each atom of the ring is carbon.

The term “nitro” means —NO₂; the term “halogen” designates —F, —Cl, —Bror —I; the term “sulfhydryl” means —SH; the term “hydroxyl” means —OH;and the term “sulfonyl” means —SO₂—.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that may berepresented by the general formulas:

wherein R₅₀, R₅₁ and R₅₂ each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R₆₁, or R₅₀ and R₅₁, taken together withthe N atom to which they are attached complete a heterocycle having from4 to 8 atoms in the ring structure; R₆, represents an aryl, acycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zeroor an integer in the range of 1 to 8. In certain embodiments, only oneof R₅₀ or R₅₁ may be a carbonyl, e.g., R₅₀, R₅₁ and the nitrogentogether do not form an imide. In other embodiments, R₅₀ and R₅₁ (andoptionally R₅₂) each independently represent a hydrogen, an alkyl, analkenyl, or —(CH₂)_(m)—R₆₁. Thus, the term “alkylamine” includes anamine group, as defined above, having a substituted or unsubstitutedalkyl attached thereto, i.e., at least one of R₅₀ and R₅₁ is an alkylgroup.

The term “acylamino” is art-recognized and refers to a moiety that maybe represented by the general formula:

wherein R₅₀ is as defined above, and R₅₄ represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R₆₁, where m and R₆₁ are as definedabove.

The term “amido” is art recognized as an amino-substituted carbonyl andincludes a moiety that may be represented by the general formula:

wherein R₅₀ and R₅₁ are as defined above. Certain embodiments of theamide in the present invention will not include imides which may beunstable.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In certain embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R₆₁, wherein m and R₆₁ are defined above.Representative alkylthio groups include methylthio, ethyl thio, and thelike.

The term “carbonyl” is art recognized and includes such moieties as maybe represented by the general formulas:

wherein X₅₀ is a bond or represents an oxygen or a sulfur, and R₅₅represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₆₁ or apharmaceutically acceptable salt, R₅₆ represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R₆₁ where m and R₆₁ are defined above. WhereX₅₀ is an oxygen and R₅₅ or R₅₆ is not hydrogen, the formula representsan “ester”. Where X₅₀ is an oxygen, and R₅₆ is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR₅₆ is a hydrogen, the formula represents a “carboxylic acid”. Where X₅₀is an oxygen, and R₅₅ is hydrogen, the formula represents a “formate”.In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiocarbonyl” group. Where X₅₀ is asulfur and R₅₅ or R₅₆ is not hydrogen, the formula represents a“thioester.” Where X₅₀ is a sulfur and R₅₆ is hydrogen, the formularepresents a “thiocarboxylic acid.” Where X₅₀ is a sulfur and R₅₅ ishydrogen, the formula represents a “thioformate.” On the other hand,where X₅₀ is a bond, and R₅₅ is not hydrogen, the above formularepresents a “ketone” group. Where X₅₀ is a bond, and R₅₅ is hydrogen,the above formula represents an “aldehyde” group.

The terms “alkoxyl” or “alkoxy” refers to an alkyl group, as definedabove, having an oxygen radical attached thereto. Representative alkoxylgroups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An“ether” is two hydrocarbons covalently linked by an oxygen. Accordingly,the substituent of an alkyl that renders that alkyl an ether is orresembles an alkoxyl, such as may be represented by one of —O-alkyl,—O-alkenyl, —O-alkynyl, —O—(CH₂)_(m)—R₆₁, where m and R₆₁ are describedabove.

The term “sulfonate” is art recognized and includes a moiety that may berepresented by the general formula:

in which R₅₇ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The term “sulfate” is art recognized and includes a moiety that may berepresented by the general formula:

in which R₅₇ is as defined above.

The term “sulfonamido” is art recognized and includes a moiety that maybe represented by the general formula:

in which R₅₀ and R₅₆ are as defined above.

The term “sulfamoyl” is art-recognized and includes a moiety that may berepresented by the general formula:

in which R₅₀ and R₅₁ are as defined above.

The term “sulfonyl” refers to a moiety that may be represented by thegeneral formula:

in which R₅₈ is one of the following: hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, heterocyclyl, aryl or heteroaryl.

The term “sulfoxido” refers to a moiety that may be represented by thegeneral formula:

in which R₅₈ is defined above.

Analogous substitutions may be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

The definition of each expression, e.g. alkyl, m, n, p, etc., when itoccurs more than once in any structure, is intended to be independent ofits definition elsewhere in the same structure.

A “selenoalkyl” refers to an alkyl group having a substituted selenogroup attached thereto. Exemplary “selenoethers” which may besubstituted on the alkyl are selected from one of —Se-alkyl,—Se-alkenyl, —Se-alkynyl, and —Se—(CH₂)_(m)—R₆₁, m and R₆₁ being definedabove.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized andrefer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl,and nonafluorobutanesulfonyl groups, respectively. The terms triflate,tosylate, mesylate, and nonaflate are art-recognized and refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl,ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations.

Certain monomeric subunits of the present invention may exist inparticular geometric or stereoisomeric forms. In addition, oligomers ofthe present invention may also be optically active. The presentinvention contemplates all such compounds, including cis- andtrans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers,(L)-isomers, the racemic mixtures thereof, and other mixtures thereof,as falling within the scope of the invention. Additional asymmetriccarbon atoms may be present in a substituent such as an alkyl group. Allsuch isomers, as well as mixtures thereof, are intended to be includedin this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction.

The term “substituted” is also contemplated to include all permissiblesubstituents of organic compounds. In a broad aspect, the permissiblesubstituents include acyclic and cyclic, branched and unbranched,carbocyclic and heterocyclic, aromatic and nonaromatic substituents oforganic compounds. Illustrative substituents include, for example, thosedescribed herein above. The permissible substituents may be one or moreand the same or different for appropriate organic compounds. Forpurposes of this invention, the heteroatoms such as nitrogen may havehydrogen substituents and/or any permissible substituents of organiccompounds described herein which satisfy the valences of theheteroatoms. This invention is not intended to be limited in any mannerby the permissible substituents of organic compounds.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Alsofor purposes of this invention, the term “hydrocarbon” is contemplatedto include all permissible compounds having at least one hydrogen andone carbon atom. In a broad aspect, the permissible hydrocarbons includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic organic compounds that may besubstituted or unsubstituted.

The phrase “protecting group” includes temporary substituents thatprotect a potentially reactive functional group from undesired chemicaltransformations. Examples of such protecting groups include esters ofcarboxylic acids, silyl ethers of alcohols, and acetals and ketals ofaldehydes and ketones, respectively. The field of protecting groupchemistry has been reviewed. Greene et al., Protective Groups in OrganicSynthesis 2^(nd) ed., Wiley, New York, (1991).

The term “electron-withdrawing group” is recognized in the art, anddenotes the tendency of a substituent to attract valence electrons fromneighboring atoms, i.e., the substituent is electronegative with respectto neighboring atoms. A quantification of the level ofelectron-withdrawing capability is given by the Hammett sigma (σ)constant. This well known constant is described in many references, forinstance, March, Advanced Organic Chemistry 251-59, McGraw Hill BookCompany, New York, (1977). The Hammett constant values are generallynegative for electron donating groups (σ(P)=−0.66 for NH₂) and positivefor electron withdrawing groups (σ(P)=0.78 for a nitro group), σ(P)indicating para substitution. Exemplary electron-withdrawing groupsinclude nitro, acyl, formyl, sulfonyl, trifluoromethyl, cyano, chloride,and the like. Exemplary electron-donating groups include amino, methoxy,and the like.

Contemplated equivalents of the oligomers, subunits and othercompositions described above include such materials which otherwisecorrespond thereto, and which have the same general properties thereof(e.g., biocompatible, antineoplastic), wherein one or more simplevariations of substituents are made which do not adversely affect theefficacy of such molecule to achieve its intended purpose. In general,the compounds of the present invention may be prepared by the methodsillustrated in the general reaction schemes as, for example, describedbelow, or by modifications thereof, using readily available startingmaterials, reagents and conventional synthesis procedures. In thesereactions, it is also possible to make use of variants that are inthemselves known, but are not mentioned here.

III. Compounds of the Present Invention

The present invention provides peptidomimetic compounds that maysuppress an immune response, e.g., by inhibiting class II MHC-mediatedactivation of T cells. For example, suitable peptidomimetics includecompounds having a structure of Formula I:

-   -   wherein, as valence and stability permit,    -   A is absent or represents a sequence of from one to four amino        acid or amino acid analog residues, preferably is absent;    -   B represents a sequence of from two to eight amino acid or amino        acid analog residues, preferably from two to six amino acid or        amino acid analog residues;    -   X is absent or represents O, S, or NR;    -   W represents a terminating group, such as OR₇ or NR₈R₉;    -   V, independently for each occurrence, represents C═O, C═S, or        SO₂;    -   R, independently for each occurrence, represents H or lower        alkyl, preferably H;    -   R₁ represents a substituted or unsubstituted alkyl, heteroalkyl,        alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl,        cycloalkyl, cycloalkylalkyl, heterocyclyl, or heterocyclylalkyl        moiety, preferably a hydrophobic moiety, most preferably        comprising from 1 to 8 carbon atoms;    -   R₂ represents a substituted or unsubstituted alkyl, heteroalkyl,        alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl,        cycloalkyl, cycloalkylalkyl, heterocyclyl, or heterocyclylalkyl        moiety, preferably a hydrophobic moiety, or R₂ and R, taken        together, form a ring having from 5 to 7 members, optionally        being substituted with from 1 to 5 substitutents and/or forming        a polycyclic structure with one or more other rings, such as        aryl, heterocyclyl, or carbocyclyl rings, e.g., a fused bicycle;    -   R₃ represents a substituted or unsubstituted alkyl, heteroalkyl,        alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl,        cycloalkyl, cycloalkylalkyl, heterocyclyl, or heterocyclylalkyl        moiety, preferably including a basic nitrogen atom (e.g., that        is protonated under physiological conditions and/or its        conjugate acid has a pKa in aqueous solution between 6 and 12,        preferably between 7 and 10); and    -   R₇, R and R₉ independently represent substituents selected from        H and substituted or unsubstituted alkyl, heteroalkyl, aryl,        aralkyl, heteroaralkyl, heteroaryl, cycloalkyl, cycloalkylalkyl,        heterocyclyl, and heterocyclylalkyl, or where R₈ and R₉, taken        togther, form a ring havng from 5 to 7 members, optionally being        substituted with from 1 to 5 substitutents and/or forming a        polycyclic structure with one or more other rings, such as aryl,        heterocyclyl, or carbocyclyl rings.

The present invention provides peptidomimetic compounds that maysuppress an immune response, e.g., by inhibiting class II MHC-mediatedactivation of T cells. For example, suitable peptidomimetics includecompounds having a structure of Formula II:

-   -   wherein, as valence and stability permit,    -   A is absent or represents a sequence of from one to four amino        acid or amino acid analog residues, preferably is absent;    -   B represents a sequence of from two to eight amino acid or amino        acid analog residues, preferably from two to six amino acid or        amino acid analog residues;    -   X is absent or represents O, S, or NR;    -   W represents OR₇ or NR₈R₉;    -   V, independently for each occurrence, represents C═O, C═S, or        SO₂;    -   R, independently for each occurrence, represents H or lower        alkyl, preferably H;    -   R₁ represents a substituted or unsubstituted alkyl, heteroalkyl,        alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl,        cycloalkyl, cycloalkylalkyl, heterocyclyl, or heterocyclylalkyl        moiety, preferably a hydrophobic moiety, most preferably        comprising from 1 to 8 carbon atoms;    -   R₂ represents a substituted or unsubstituted alkyl, heteroalkyl,        alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl,        cycloalkyl, cycloalkylalkyl, heterocyclyl, or heterocyclylalkyl        moiety, preferably a hydrophobic moiety, or R₂ and R, taken        together, form a ring having from 5 to 7 members, optionally        being substituted with from 1 to 5 substitutents and/or forming        a polycyclic structure with one or more other rings, such as        aryl, heterocyclyl, or carbocyclyl rings, e.g., a fused bicycle;    -   i represents an integer from 0-1, preferably 0;    -   j represents an integer from 1-2, preferably 1;    -   k represents an integer from 1-3, preferably 2;    -   R₆ is absent or represents from 1-4 substitutents on the        nitrogen-containing ring to which it is attached, selected from        substituted or unsubstituted lower alkyl, haloalkyl, halogen,        hydroxyl, and amino; and    -   R₇, R₈ and R₉ independently represent substituents selected from        H and substituted or unsubstituted alkyl, heteroalkyl, aryl,        aralkyl, heteroaralkyl, heteroaryl, cycloalkyl, cycloalkylalkyl,        heterocyclyl, and heterocyclylalkyl, or where R₈ and R₉, taken        togther, form a ring havng from 5 to 7 members, optionally being        substituted with from 1 to 5 substitutents and/or forming a        polycyclic structure with one or more other rings, such as aryl,        heterocyclyl, or carbocyclyl rings.

In certain embodiments of Formula I, R₃ represents a side-chain ofarginine or lysine or a side chain having the structure of

wherein i, j, k, and & are defined as described for Formula II. Incertain embodiments of Formula I, R₃ includes a guanidine or guanidiniummoiety, e.g., included in or attached to a ring or included in or at theterminus of a chain. In certain embodiments of Formula I, R₃ representsa cycloalkyl, alkyl, or an aminoalkyl group, such as a side-chain ofallo-isoleucine, cyclohexylglycine, citrulline, lysine, or ornithine,including N-methyl and N,N-dimethyl variants of lysine, citrulline, andornithine.

In certain embodiments of Formula I, B represents two amino acid oramino acid analog residues and W includes a terminating group asdescribed in greater detail below. Preferably, the amino acid or analogresidues are attached through secondary amide bonds (i.e., wherein thenitrogens bear a hydrogen substituent).

In certain embodiments of Formula II, R₆ is absent, and in otherembodiments, & includes a lower alkyl substituent.

In certain embodiments of Formula I and II, R₂ represents substituted orunsubstituted cycloalkyl, cycloalkylalkyl, aryl, aralkyl,

In certain embodiments of Formulae I and II, the first residue of B (theamino acid or analog residue attached to V) has a side-chain that is Hor, preferably, a C1-C8 alkyl or M1-M8 heteroalkyl (including, forexample, alanine, Acm-cysteine, Prm-cysteine, acetyl-cysteine, and Nva,e.g., C1-C6 alkyl or M1-M6 heteroalkyl), or a substituted orunsubstituted aryl, aralkyl, heteroaryl, or heteroalkyl (e.g.,methylphenyl or phenylmethyl) or the first residue of B is an amino acidanalog comprising a 5-8-membered nitrogen-containing heterocyclyl ringbearing a C═O, C═S, or SO₂ group, optionally fused to a benzene ring(e.g., Tic, azaTic, Disc, Thiq, etc.). Preferred residues at thisposition include Tic and Disc, although any residue employed at thisposition in the examples of Tables 1-3 may be present at this position.

In certain embodiments of Formulae I and II, the second residue of B(the amino acid or analog residue attached to V being the first) has aside-chain that is H or, preferably, a C1-C6 alkyl, M1-M6 heteroalkyl,or cycloalkyl, even more preferably C3-C5 alkyl or M 3-M5 heteroalkyl,either b ranched or unbranched, or cycloalkyl. Exemplary residuesinclude glycine, isoleucine, Nle, Chg, Met(O) (oxidized methionine), andalpha-aminoisobutyric acid. In certain embodiments, the second residueof B is a residue that is substantially isometric with a dipeptide, suchas on Odapdc or Haic residue (as defined below). Preferred residues atthis position include Met and Nle, although any residue employed at thisposition in the examples of Tables 1-3 may be present at this position.

In certain embodiments of Formulae I and II, R and R₂ are not takentogether to form a ring. In embodiments wherein R and R₂ taken togetherform a ring, the ring is preferably a 6- or 7-membered ring, or is asubstituted (e.g., bicyclic) 5-membered ring.

In certain embodiments of Formulae I and II, R₁XV, taken together,represent an alkanoyl, alkenoyl, aryl carbonyl, or an aminoalkanoylgroup. In certain such embodiments, the acyl group is a benzoyl group, alower alkanoyl group, or a lower aminoalkanoyl group, such as an acetyl,propanoyl, aminopropanoyl, or aminobutanoyl group.

In certain embodiments of Formulae I and II, B represents from 2 to 6amino acid or amino acid analog residues, preferably 2 to 5 amino acidor amino acid analog residues. In certain embodiments, particularlywherein B represents four or fewer amino acid or amino acid analogresidues, preferably three or fewer, W represents a terminating group.Exemplary terminating groups are depicted in Table 1 (a, b and c), andinclude nitrogen atoms (e.g., forming an amide with a terminal carboxylof B) bearing substituents selected from H, substituted andunsubstituted alkyl, aryl, aralkyl, heteroaralkyl, heteroaryl,cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl,preferably from H, substituted and unsubstituted alkyl, aryl, aralkyl,cycloalkyl, and cycloalkylalkyl. Suitable substituents include hydroxyl,ether, and amino substituents. Preferably, a terminating group includesat least six non-hydrogen atoms including the nitrogen attached to B,preferably at least eight non-hydrogen atoms. Preferably, a terminatinggroup W includes a nitrogen substituted with an aralkyl or heteroaralkylsubstituent, such as a benzyl or phenethyl substituent. In certain suchembodiments, the nitrogen bears a second substituent selected from H,lower alkyl, hydroxy-lower alkyl, and hydroxy-lower alkyl-O-lower alkyl.In certain embodiments, a terminating group W is a nitrogen-containingheterocyclyl substituent, preferably fused with an aryl or heteroarylring, attached to B through the nitrogen atom of the ring. Suchterminating groups include tetrahydroisoquinoline, indoline,isoindoline, morpholine, piperidine, etc.

Certain embodiments of Formulae I and II, such as by appropriateselection of R₁ or W, preferably R₇, R₈ or R₉, may provide a prodrugthat is converted to an active compound of the invention underphysiological conditions. For example, where W is part of an ester, theester can be cleaved under physiological conditions.

In certain embodiments of Formulae I and II, at least one of R₁, R₇, R₈or R₉ is a hydrophobic residue, preferably R₈ or R₉. In otherembodiments, the hydrophobicity of the compound, for example asestimated using the method of Meyan et al, 1995 (J. Pharm Sci.84:83-92), lies between a cLogP of around 2.0 to around 6.0, preferablybetween around 3.0 to around 6.0 most preferably between around 4.0 toaround 5.5. However, compounds that possess an estimated cLogP value ofoutside this range are contemplated by this invention, for examplecompounds having a cLogP of around 3.0 to around 4.0.

In certain embodiments of Formulae I and II, A and B together includebetween 2 and 8, e.g., between 3 and 6 amino acid or amino acid analogresidues. Preferably, A and B together include around 2 or around 5amino acid or amino acid analog residues.

The portion of Formulae I and II flanked by (but not including) B and(VCHR₂) is referred to herein as an ‘arginine-like’ residue. Inembodiments wherein i represents 0, j represents 1, and k represents 2,the arginine-like residue is referred to herein as a Gpg residue(guanylpiperidyl glycine). Such residues are known in the art, and aredescribed in PCT publication WO 00/78796 and references cited therein.In preferred embodiments, the arginine-like residue is enriched for anS-configuration at the alpha stereocenter of the amino acid, e.g.,preferably is at least 60%, 75%, 85%, 90%, or even 95% or more enrichedfor the S-enantiomer of this residue. In certain embodiments, subjectinhibitors display increased stability, e.g., have a plasma half-life atleast 1.25, 1.5, or preferably 3 times as long, preferably at least fivetimes as long, and increased binding affinity with an MHC Class IImolecule (e.g., 0401, 0101, or 0404), e.g., bind with an affinity atleast 1.25, 1.5, or preferably 3 times as great, as an analogouspeptidyl compound wherein the arginine-like residue is replaced byarginine.

Other references which describe arginine-like residues useful in thepresent invention include International Applications Nos. WO 99/61476and WO 01/27141, Jones et al., Bioorg. Med. Chem. Lett. 1999, 9,2109-2114; Cunningham et al., Bioorg. Med. Chem. Lett. 1997, 7, 19-24;Hanson et al., Bioorg. Med. Chem. Lett. 1996, 6, 1931-1936; Jones etal., Bioorg. Med. Chem. Lett. 1999, 9, 2115-2118, Tamura et al., Bioorg.Med. Chem. Lett. 2000, 10, 745-49, Falcioni et al., 1999, Nature Biotech17, 562-567, and Schmidt et al., Proc. Am. Pept. Symp., 16^(th) (2000),Meeting Date 1999, 634-635.

In certain embodiments of Formula I and Formula II, the amino acids of Aand/or B include a transcellular polypeptide sequence, such as aredescribed in U.S. Pat. No. 6,495,526. The transcellular polypeptidesequence can be an internalizing peptide, such as may be derived from apolypeptide selected from antepennepedia protein, HIV transactivating(TAT) protein, mastoparan, melittin, bombolittin, delta hemolysin,pardaxin, Pseudomonas exotoxin A, clathrin, Diphtheria toxin and C9complement protein, or a fragment thereof.

In one embodiment, the internalizing peptide is derived from thedrosophila antepennepedia protein, or homologs thereof. The 60 aminoacid long long homeodomain of the homeo-protein antepennepedia has beendemonstrated to translocate through biological membranes and canfacilitate the translocation of heterologous polypeptides to which it iscoupled. See for example Derossi et al. (1994) J Biol Chem269:10444-10450; and Perez et al. (1992) J Cell Sci 102:717-722.Recently, it has been demonstrated that fragments as small as 16 aminoacids long of this protein are sufficient to drive internalization. SeeDerossi et al. (1996) J Biol Chem 271:18188-18193. The present inventioncontemplates coupling at least a portion of the antepernepedia protein(or homolog thereof) to a peptide or peptidomimetic of Formula I or IIto increase the transmembrane transport of the compound, relative to thecompound alone, by a statistically significant amount.

Another example of an internalizing peptide is the HIV transactivator(TAT) protein. This protein appears to be divided into four domains(Kuppuswamy et al. (1989) Nucl Acids Res. 17:3551-3561). Purified TATprotein is taken up by cells in tissue culture (Frankel and Pabo, (1989)Cell 55:1189-1193), and peptides, such as the fragment corresponding toresidues 37-62 of TAT, are rapidly taken up by cell in vitro (Green andLoewenstein, (1989) Cell 55:1179-1188). The highly basic region mediatesinternalization and targeting of the internalizing moiety to the nucleus(Ruben et al., (1989) J. Virol. 63:1-8). Peptides or analogs thatinclude a sequence present in the highly basic region, such asCFITKALGISYGRKKRRQRRRPPQGS, or a sub sequence thereof such asYGRKKRRQRRR, can be conjugated to compounds of Formula I or II to aid ininternalization and targeting those compounds to the intracellularmilieu.

In certain such embodiments, the amino acid sequence of A or B is longerthan defined with respect to Formula I or II to permit attachment of anamino acid sequence of sufficient length to promote internalization ofthe compound.

Particularly preferred compounds of the invention are set forth below asP53, P74, P101, P102 and P69, most preferably P69 (see Table 1a).

The compounds of the invention can also serve as lead compounds for thedevelopment of analog compounds. The analogs should have a stabilizedelectronic configuration and molecular conformation that allows keyfunctional groups to be presented to for example MHC class II protein insubstantially the same way as the lead compound. In particular, theanalog compounds have spatial electronic properties which are comparableto the binding region, but can be larger or smaller molecules than thelead compound. Identification of analog compounds can be performedthrough use of techniques such as self-consistent field (SCF) analysis,configuration interaction (CI) analysis, and normal mode dynamicsanalysis. Thus, the compounds of the present invention can be furthermodified as a lead compound to achieve

-   -   (h) modified site of action, spectrum of activity, organ        specificity, and/or    -   (i) improved potency, and/or    -   (j) decreased toxicity (improved therapeutic index), and/or    -   (k) decreased side effects, and/or    -   (l) modified onset of therapeutic action, duration of effect,        and/or    -   (m) modified pharmakinetic parameters (resorption, distribution,        metabolism and excretion), and/or    -   (n) modified physico-chemical parameters (solubility,        hygroscopicity, color, taste, odor, stability, state), and/or    -   (O) improved general specificity, organ/tissue specificity,        and/or    -   (p) optimized application form and route by    -   (q) esterification of carboxyl groups, or    -   (r) esterification of hydroxyl groups with carbon acids, or    -   (s) esterification of hydroxyl groups to, e.g. phosphates,        pyrophosphates or sulfates or hemi succinates, or    -   (t) formation of pharmaceutically acceptable salts, or    -   (u) formation of pharmaceutically acceptable complexes, or    -   (v) synthesis of pharmacologically active polymers, or    -   (w) introduction of hydrophylic moieties, or    -   (x) introduction/exchange of substituents on aromates or side        chains, change of substituent pattern, or    -   (y) modification by introduction of isosteric or bioisosteric        moieties, or    -   (z) synthesis of homologous compounds, or    -   (aa) introduction of branched side chains, or    -   (bb) conversion of alkyl substituents to cyclic analogues, or    -   (cc) derivatisation of hydroxyl group to ketales, acetales, or    -   (dd) N-acetylation to amides, phenylcarbamates, or    -   (ee) synthesis of Mannich bases, imines, or    -   (ff) transformation of ketones or aldehydes to Schiffs bases,        oximes, acetales, ketales, enolesters, oxazolidines,        thiozolidines    -   or combinations of any one thereof. The various steps recited        above are generally known in the art. For example, computer        programs for implementing these techniques are available; e.g.,        Rein, Computer-Assisted Modeling of Receptor-Ligand Interactions        (Alan Liss, New York, 1989). Methods for the preparation of        chemical derivatives and analogues are well known to those        skilled in the art and are described in, for example, Beilstein,        Handbook of Organic Chemistry, Springer edition New York Inc.,        175 Fiftih Avenue, New York, N.Y. 10010 U.S.A. and Organic        Synthesis, Wiley, New York, USA. Furthermore, peptide mimetics        and/or computer aided design of appropriate derivatives and        analogues can be used, for example, according to the methods        described above. Methods for the lead generation in drug        discovery also include using proteins and detection methods such        as mass spectrometry (Cheng et al. J. Am. Chem. Soc. 117 (1995),        8859-8860) and some nuclear magnetic resonance (NMR) methods        (Fejzo et al., Chem. Biol. 6 (1999), 755-769; Lin et al., J.        Org. Chem. 62 (1997), 8930-8931). They may also include or rely        on quantitative structure-action relationship (QSAR) analyses        (Kubinyi, J. Med. Chem. 41 (1993), 2553-2564, Kubinyi, Pharm.        Unserer Zeit 23 (1994), 281-290) combinatorial biochemistry,        classical chemistry and others (see, for example, Holzgrabe and        Bechtold, Pharm. Acta Helv. 74 (2000), 149-155).

The present invention further relates to therapeutic preparationscomprising a subject compound and an excipient, such as apharmaceutically acceptable or sterile excipient. The invention furtherrelates to a method for treating or preventing a condition characterizedby MHC-II-mediated activation of T cells, comprising administering to ananimal, such as a human, a composition comprising a compound as setforth above. The invention further relates to uses of a subject compoundfor the preparation of a pharmaceutical composition. Such pharmaceuticalcomposition may be suitable for the treatment or prevention of acondition characterized by MHC-II-mediated activation of T cells. Incertain embodiments, the condition is an autoimmune disorder, e.g.,rheumatoid arthritis or multiple sclerosis.

In certain embodiments, a subject inhibitor is selective for onetherapeutic isotype or allotype, such as HLA-DR or DRB1*0101, over asecond isotype or allotype, or over most other isotypes or allotypes.Thus, a subject inhibitor may have an ED₅₀ at least 5 or 10 times lowerfor one isotype or allotype, preferably at least 100 times lower, evenmore preferably at least 1000 times lower, over one or more other HLAisotypes or allotypes. Similarly, a subject inhibitor may have an IC₅₀at least 5 or 10 times lower for one isotype or allotype, preferably atleast 100 times lower, even more preferably at least 1000 times lower,over one or more other HLA isotypes or allotypes.

In certain embodiments, the invention provides a method of conducting apharmaceutical business by selecting one or more compounds as disclosedherein for their ability to bind to MHC class II protein, conductingtherapeutic profiling of said compound for efficacy and toxicity inanimals, preparing a package insert describing the use of said compoundfor suppressing an immune response, and marketing the multivalentcomposition for suppressing an immune response. The invention alsoprovides a kit comprising a compound as disclosed herein andinstructions for administering the compound to suppress an immuneresponse.

In another embodiment, the invention provides a method of conducting alife science business by selecting one or more compounds as describedherein for their ability to bind to MEC class II protein, and licensing,jointly developing, or selling to a third party, the rights formanufacturing, marketing, selling or using said compound for suppressingan immune response.

IV. Therapeutic Applications

The subject compounds can be utilized for a wide range of medicaltreatments. For example, subject compounds may be employed inconjunction with solid organ transplants. Preferably, the organ isselected from the group consisting of heart, liver, kidney, adrenalcortex, lung, intestine, pancreas, cornea and skin. Most preferably, thetarget organ is selected from the group consisting of heart, kidney,liver, cornea, and skin. For example, a patient may be treated with asubject compound before or after receiving a transplant or allograft toprevent or ameliorate immune reactions that might lead to rejection ofthe transplant or graft vs. host disease. Sustained releases of thesubject compounds, e.g., from a biodegradable polymer implant, or frombiodegradable polymeric microparticles or nanoparticles, are alsocontemplated.

The compounds of the invention are also useful in treating diseases ofthe immune system characterized by unwanted, dysfunctional, or aberrantactivation of T cells by MHC class II polypeptides. Such immune diseasesinclude, but are not limited to, rheumatoid arthritis, juvenilearthritis, multiple sclerosis, Grave's disease, insulin-dependentdiabetes, narcolepsy, psoriasis, systemic lupus erythematosus,ankylosing spondylitis, allograft rejection, Hashimoto's disease,myasthenia gravis, pemphigus vulgaris, thyroiditis, glomeruloneplritis,insulitis, irritable bowel disease, pancreatitis, and primary biliarycirrhosis. Other disorders for which the compounds of the invention maybe employed to relieve the symptoms of, treat or prevent the occurrenceor reoccurrence of include, for example, Sjogren syndrome, scleroderma,polymyositis, dermatomyositis, bullous pemphigoid, Goodpasture'ssyndrome, autoimmune hemolytic anemia, pernicious anemia, idiopathicthrombocytopenic purpura, and Addison's disease, and the like. For suchtreatments, the compounds described herein may be administered in anamount sufficient to inhibit MHC-II mediated T cell activation by atherapeutically acceptable amount.

Specific autoimmune dysfunctions are often correlated with specific MHCtypes. DQ/DR haplotypes in humans and their associations with autoimmunediseases are well known, as described in U.S. Pat. No. 6,045,796. Incertain embodiments, it may be advantageous to determine the genotypeand/or phenotype of a patient to be treated with a subject inhibitor,e.g., to select a drug suitable for treating a disease or conditionassociated with the patient's haplotype, or to determine a patient'sgenotype and/or phenotype, as appropriate, for the selection and/orprescription of a particular drug. In a preferred embodiment, theassociation between a disease and specific MHC types is so strong thatdetermining the genotype and/or phenotype of a patient may not berequired. Methods for determining the haplotype of an animal, such as ahuman, are well known in the art, and any suitable technique may be usedto make such a determination, for example, by analyzing DNA restrictionfragment length polymorphism (RFLP) using DNA probes that are specificfor the MHC locus being examined. Methods of preparing probes for theMHC loci are known to those skilled in the art. See, for example,Gregersen et al., (1986), Proc. Natl. Acad. Sci. U.S.A. 79:5966, whichis incorporated herein by reference. The patient's haplotype may then becompared with haplotypes with known disease associations. As an example,over 90% of rheumatoid arthritis patients have a haplotype of DR4(Dw4),DR4(Dw14), or DR1. In particular, juvenile rheumatoid arthritis (e.g.,pauciarticular juvenile rheumatoid arthritis) is associated withHLA-DPB2.1 (Begovich et al., 1989, PNAS 86:9489-9493). Approximately 70%of patients with insulin-dependent diabetes mellitus express HLA-DQ3.2B,DQA1, or DQB1, and susceptibility to the autoimmune dermatologic diseasepemphigus vulgaris is linked to expression of HLA-DQB1.3 (Scharf et al.,1989, PNAS 86:6215-6219). Allergic reactions to ragweed are known to beassociated with DR2 alleles. Marsh et al., (1989) Cold Spring Harb SympQuant Biol 54:459-70, which is incorporated herein by reference.

Methods for In Vitro Testing

The biological activity of the inhibitor, e.g., the ability to inhibitantigen-specific T cell activation, may be assayed in a variety ofsystems. In one method, purified class II MHC molecules are incorporatedinto phospholipid vesicles by detergent dialysis. The resultant vesiclesare then allowed to fuse to clean glass cover slips to produce on each aplanar lipid bilayer containing MHC molecules (Brian and McConnell,Proc. Natl. Acad. Sci. USA (1984) 81: 6159). The inhibitors to be testedare detectably labeled and then incubated on the plates with purifiedMHC proteins which have been formulated into lipid membrane bilayers.Inhibitors that bind to the MHC molecules are identified by detectinglabel bound to the plate.

In a second exemplary protocol, an excess of inhibitor is incubated withan antigen-presenting cell expressing an MHC allotype of interest,(e.g., a DR of interest) and a T cell clone which recognizes a selectedpeptide (e.g., tetanus toxin 830-843) and MHC molecule (e.g., the DR ofinterest), and the antigenic peptide itself. The assay culture isincubated for a sufficient time for T cell proliferation, such as fourdays, and proliferation is then measured using standard procedures, suchas pulsing with tritiated thymidine during the last 18 hours ofincubation. The percent inhibition, compared to controls which receivedno inhibitor, is then calculated.

A third protocol is described in U.S. Pat. No. 5,736,507. In thatdisclosure, the peptide binding studies were performed using an improvedversion of a semi-quantitative binding assay described previously(Joosten et al., Int. Immunol. 6:751, 1994). Adapted for the presentinvention, purified MHC molecules (0.5-500 nM) may be incubated atpH=5.0 with 50 nM biotinylated indicator peptide and a concentrationrange of inhibitor in a final volume of 25 μl binding buffer (e.g., PBS,1 mM AEBSF, 1 mM N-ethyl maleimide, 8 mM EDTA, 10 μM pepstatin A, 0.01%NaN₃, 0.05% NP-40 and 5% DMSO) may be employed.

After approximately 45 hours incubation at room temperature, bound andunbound indicator peptides may be separated either by SDS-PAGE incombination with blotting on a nitrocellulose filter (BioRad) or byvacuum DOT blotting using a nitrocellulose filter (BioRad) and 96 wellsHybry Dot equipment (BRL). Blots may be blocked with 0.5% DNA blockingreagent (Boehringer Mannheim, Germany) in 0.1 M maleic acid pH=7.5, 150mM NaCl. After ½ hour, blots are washed in PBS, 0.02% Tween 20 (Sigma,St. Louis, USA) and incubated with Streptavidin-HRPO (SouthernBiotechnology) in a 1:40,000 or 1:5,000 dilution respectively. DR-bound,biotinylated indicator peptide is detected by enhanced chemoluminescenceusing a Western Blot ECL kit (Amersham, U.K.) according to themanufacturer's instructions. Preflashed films (hyperfilm-ECL, Amersham,U.K.) are exposed for 10 minutes. The relative binding affinity of agiven peptide is related to competition with the indicator peptide. Thisrelative affinity is defined as the inhibitor concentration at which thesignal is reduced to 50% (^(R)IC₅₀).

A similar protocol, detailed in the Exemplification below, is based onthe protocol taught by Siklodi et al., Human Immunology, 59 (1998)463-471, and employs competitive binding of subject inhibitors. Anysuitable MHC-II allotype may be employed in such assays, and, asdescribed below, the method is suitable for screening libraries ofcompounds for their ability to bind MHC-II molecules.

Other suitable methods to determine the in-vitro biological activity ofan inhibitor may be taken from the examples below.

Model Systems for In Vivo Testing

The capacity of compounds to inhibit antigen presentation in an in vitroassay has been correlated to the capacity of the compounds to inhibit animmune response in vivo. In vivo activity may be determined in animalmodels, for example, by administering an antigen known to be restrictedto the particular MHC molecule of interest, together with a testinhibitor of the present invention. T lymphocytes are subsequentlyremoved from the animal and cultured with a dose range of antigen.Inhibition of stimulation is measured by conventional means, e.g.,pulsing with ³H-thymidine, and comparing to appropriate controls.Preferably, as described in the Exemplification below, an animal modelwill be genetically modified to express a human MHC class II allotype ofinterest in place of endogenous MHC class II molecules. Certainexperimental details will of course be apparent to the skilled artisan.See also, Adorini, et al., Nature 334:623-625 (1988), and Ito et al.(1996) J. Exp. Med. 183:2635-2644, both incorporated herein byreference.

The following are exemplary model systems for diseases of the immunesystem, which can be used to evaluate the effects of the compounds ofthe invention on these conditions. A skilled artisan would be able, withno more than routine experimentation or research, to identify othermodels suitable for testing compounds of the invention against these andother diseases of the immune system.

Experimental autoimmune encephalomyelitis (EAE) is a model for multiplesclerosis (MS) that induced by immunization with a myelin protein, e.g.,myelin basic protein (MBP), proteolipid protein (PP) or mouseoligodendrocyte glycoprotein (MOG), in mice transgenic for anMS-associated human class II allotype and deficient of mouse class IImolecules as described by Ito et al. (1996).

Collagen induced arthritis (CIA) is a model for rheumatoid arthritis(RA), induced by immunization with type II collagen in mice transgenicfor an RA-associated human class II molecule (Rosloniec et al., J. Exp.Med. 185: 1113 (1997), & J. Immunol. 160: 2573-2578, (1998)).

V. Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically effectiveamount of one or more compounds of the subject invention, such asdescribed above, formulated together with one or more pharmaceuticallyacceptable carriers (additives) and/or diluents for use in the treatmentof aberrant T cell activation or an autoimmune disease, for example,rheumatoid arthritis or multiple sclerosis. As described in detailbelow, the pharmaceutical compositions of the present invention may bespecially formulated for administration in solid or liquid form,including those adapted for the following: (1) oral administration, forexample, drenches (aqueous or non-aqueous solutions or suspensions),tablets, capsules, boluses, powders, granules, pastes for application tothe tongue; (2) parenteral administration, for example, by subcutaneous,intramuscular or intravenous injection as, for example, a sterilesolution or suspension; (3) topical application, for example, as acream, ointment or spray applied to the skin; or (4) intravaginally orintrarectally, for example, as a pessary, cream, foam, or suppository.In certain embodiments, the pharmaceutical preparations may benon-pyrogenic, i.e., do not elevate the body temperature of a patient.

The phrase “therapeutically effective amount” as used herein means thatamount of a compound, material, or composition comprising an inhibitorof the subject invention which is effective for producing some desiredtherapeutic effect. Such therapeutic effect may result from, forexample, inhibition of unwanted T cell activation.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject compoundsfrom one organ, or portion of the body, to another organ, or portion ofthe body. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

As set out above, certain embodiments of the present subject compoundsmay contain a basic functional group, such as amino or alkylamino, andare, thus, capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable acids. The term “pharmaceutically acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of such inhibitors of MHC activity.These salts can be prepared in situ during the final isolation andpurification of the compounds of the present invention, or by separatelyreacting a purified compound of the invention in its free base form witha suitable organic or inorganic acid, and isolating the salt thusformed. Representative salts include the hydrobromide, hydrochloride,sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate,palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.(See, for example, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm.Sci. 66:1-19)

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of an inhibitor of an MHC activity such as T cell activation.These salts can likewise be prepared in situ during the final isolationand purification of the compounds of the present invention, or byseparately reacting the purified compound in its free acid form with asuitable base, such as the hydroxide, carbonate or bicarbonate of apharmaceutically acceptable metal cation, with ammonia, or with apharmaceutically acceptable organic primary, secondary or tertiaryamine. Representative alkali or alkaline earth salts include thelithium, sodium, potassium, calcium, magnesium, and aluminum salts andthe like. Representative organic amines useful for the formation of baseaddition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like. (See, forexample, Berge et al., supra)

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient that can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of inhibitor which produces a therapeutic effect.Generally, out of one hundred percent, this amount will range from about1 percent to about ninety-nine percent of active ingredient, preferablyfrom about 5 percent to about 70 percent, most preferably from about 10percent to about 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association an inhibitor of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. An inhibitor of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered inhibitormoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulations so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract such as the small or large intestines,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes. The activeingredient can also be in micro-encapsulated form, if appropriate, withone or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active inhibitor(s) of the presentinvention, may contain suspending agents as, for example, ethoxylatedisostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agarand tragacanth, and mixtures thereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active inhibitor.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive inhibitor, excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Such dosageforms can be made by dissolving or dispersing the inhibitor of thepresent invention in the proper medium. Absorption enhancers can also beused to increase the flux of the drug across the skin. The rate of suchflux can be controlled by either providing a rate controlling membraneor dispersing the compound of the present invention in a polymer matrixor gel.

Opthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more inhibitors of the invention incombination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and other antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the therapeutic effect of aninhibitor, it is desirable to slow the absorption of the inhibitor fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material havingpoor water solubility. The rate of absorption of the inhibitor thendepends upon its rate of dissolution which, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered inhibitor form is accomplished by dissolvingor suspending the inhibitor in an oil vehicle.

Injectable depot forms are made by forming microencapsuled matrices ofthe subject inhibitors in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

In certain embodiments, a compound as described herein is administeredconjointly with another therapeutic agent, e.g., anotherimmunosuppressant agent, an agent or substance that triggers an unwantedimmune response (for example, transplanted cells), or an agent that actstogether with the immunosuppressant to achieve a desired therapeuticeffect, such as an antiiflammatory agent. For example, the compound andagent or substance can be administered in a single composition such as atablet, in separate compositions simultaneously, or in separatecompositions at different times as part of a therapeutic regimen, etc.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given by formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administration is preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrastemal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

Regardless of the route of administration selected, the inhibitorsuseful in the subject method may be used in a suitable hydrated form,and/or the pharmaceutical compositions of the present invention, areformulated into pharmaceutically acceptable dosage forms by conventionalmethods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response, e.g., amelioration of symptoms of rheumatoidarthritis or multiple sclerosis, for a particular patient, composition,and mode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular inhibitor employed, or theester, salt or derivative thereof, the route of administration, the timeof administration, the rate of excretion of the particular compoundbeing employed, the duration of the treatment, other drugs, compoundsand/or materials used in combination with the particular inhibitoremployed, the age, sex, weight, condition, general health and priormedical history of the patient being treated, and like factors wellknown in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a potent inhibitor, e.g., having anEC₅₀ in the range of 1 mM to sub-nanomolar, will be that amount of thecompound which is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above. Generally, intravenous, intracerebroventricular andsubcutaneous doses of the compounds of this invention for a patient,when used for the indicated effects, will range from about 0.0001 toabout 1000 mg per kilogram of body weight per day, though preferably 0.5to 300 mg per kilogram.

If desired, the effective daily dose of the active inhibitor may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

In a preferred embodiment, the inhibitor agent is formulated for oraladministration, as for example in the form of a solid tablet, pill,capsule, caplet or the like (collectively hereinafter “tablet”) or anaqueous solution or suspension. In a preferred embodiment of the tabletform of the inhibitor agent, the tablets are preferably formulated suchthat the amount of inhibitor agent (or inhibitor agents) provided in 20tablets, if taken together, would provide a dose of at least the medianeffective dose (ED₅₀), e.g., the dose at which at least 50% ofindividuals exhibited a therapeutic affect. For example, for aninhibitor agent, the therapeutic effect would be a quantal effect ofinhibition of MHC class II molecule-mediated T cell activation (e.g., astatistically significant reduction in inflammation). More preferably,the tablets are formulated such that the total amount of inhibitor agent(or inhibitor agents) provided in 10, 5, 2 or 1 tablets would provide atleast an ED₅₀ dose to a patient (human or non-human mammal). In otherembodiments, the amount of inhibitor agent (or inhibitor agents)provided in 20, 10, 5 or 2 tablets taken in a 24 hour time period wouldprovide a dosage regimen providing, on average, a mean plasma level ofthe inhibitor agent(s) of at least the ED₅₀ concentration (theconcentration for 50% of maximal effect of, e.g., inhibiting an MHCactivity), though preferably less than 100 times the ED₅₀, and even morepreferably less than 10 or 5 times the ED₅₀. In preferred embodiments, asingle dose of tablets (1-20 tablets) provides about 0.25 mg to 1250 mgof an inhibitor agent(s).

Likewise, the inhibitor agents can be formulated for parenteraladministration, as for example, for subcutaneous, intramuscular orintravenous injection, e.g., the inhibitor agent can be provided in asterile solution or suspension (collectively hereinafter “injectablesolution”). The injectable solution is preferably formulated such thatthe amount of inhibitor agent (or agents) provided in a 200 cc bolusinjection would provide a dose of at least the median effective dose,though preferably less than 100 times the ED₅₀, and even more preferablyless than 10 or 5 times the ED₅₀. More preferably, the injectablesolution is formulated such that the total amount of inhibitor agent (oragents) provided in 100, 50, 25, 10, 5, 2.5, or 1 cc injections wouldprovide an ED₅₀ dose to a patient, and preferably less than 100 timesthe ED₅₀, and even more preferably less than 10 or 5 times the ED₅₀. Inother embodiments, the amount of inhibitor agent (or inhibitor agents)provided in a total volume of 100 cc, 50, 25, 5 or 2 cc to be injectedat least twice in a 24 hour time period would provide a dosage regimenproviding, on average, a mean plasma level of the inhibitor agent(s) ofat least the ED₅₀ concentration, though preferably less than 100 timesthe ED₅₀, and even more preferably less than 10 or 5 times the ED₅₀. Inpreferred embodiments, a single dose injection provides about 0.25 mg to1250 mg of inhibitor agent.

For continuous intravenous infusion, e.g., drip or push, the inhibitoragent may be provided in a sterile dilute solution or suspension(collectively hereinafter “i.v. injectable solution”). The i.v.injectable solution is preferably formulated such that the amount ofinhibitor agent (or inhibitor agents) provided in a 1 L solution wouldprovide a dose, if administered over 15 minutes or less, of at least themedian effective dose, though preferably less than 100 times the ED₅₀,and even more preferably less than 10 or 5 times the ED₅₀. Morepreferably, the i.v. injectable solution is formulated such that thetotal amount of inhibitor agent (or inhibitor agents) provided in 1 Lsolution administered over 60, 90, 120 or 240 minutes would provide anED₅₀ dose to a patient, though preferably less than 100 times the ED₅₀,and even more preferably less than 10 or 5 times the ED₅₀. In preferredembodiments, a single i.v. “bag” provides about 0.25 mg to 5000 mg ofinhibitor agent per liter i.v. solution, more preferably 0.25 mg to 2500mg, and even more preferably 0.25 mg to 1250 mg.

An ED₅₀ dose, for a human, is based on a body weight of from 2 Kg to 125Kg, though more preferably for an adult in the range of 50 to 125 Kg.

Potential inhibitors may be assessed for ED₅₀ values for any inhibition,including for example therapeutic activity towards rheumatoid arthritisor multiple sclerosis, using any of a number of well known techniques inthe art, such as those described above.

VI. Combinatorial Synthesis of Subject Inhibitors

The compounds of the present invention, particularly libraries ofvariants having various representative classes of substituents, areamenable to combinatorial chemistry and other parallel synthesis schemes(see, for example, PCT WO 94/08051). The result is that large librariesof related compounds, e.g., a variegated library of compoundsrepresented by formula I or II above, can be screened rapidly in highthroughput assays in order to identify potential lead compounds, as wellas to refine the specificity, toxicity, and/or cytotoxic-kinetic profileof a lead compound, e.g., by using one of the assays described herein.

Simply for illustration, a combinatorial library for the purposes of thepresent invention is a mixture of chemically related compounds which maybe screened together for a desired property. The preparation of manyrelated compounds in a single reaction greatly reduces and simplifiesthe number of screening processes which need to be carried out.Screening for the appropriate physical properties can be done byconventional methods.

Diversity in the library can be created at a variety of differentlevels. For instance, the substrate aryl groups used in thecombinatorial reactions can be diverse in terms of the core aryl moiety,e.g., a variegation in terms of the ring structure, and/or can be variedwith respect to the other substituents.

A variety of techniques are available in the art for generatingcombinatorial libraries of small organic molecules such as the subjectinhibitors. See, for example, Blondelle et al. (1995) Trends Anal. Chem.14:83; the Affymax U.S. Pat. Nos. 5,359,115 and 5,362,899: the EllmanU.S. Pat. No. 5,288,514: the Still et al. PCT publication WO 94/08051;Chen et al. (1994) JACS 116:2661: Kerr et al. (1993) JACS 115:252; PCTpublications WO92/10092, WO93/09668 and WO91/07087; and the Lerner etal. PCT publication WO93/20242). Accordingly, a variety of libraries onthe order of about 100 to 1,000,000 or more diversomers of the subjectinhibitors can be synthesized and screened for particular activity orproperty.

A) Direct Characterization

A growing trend in the field of combinatorial chemistry is to exploitthe sensitivity of techniques such as mass spectrometry (MS), forexample, which can be used to characterize sub-femtomolar amounts of acompound, and to directly determine the chemical constitution of acompound selected from a combinatorial library. For instance, where thelibrary is provided on an insoluble support matrix, discrete populationsof compounds can be first released from the support and characterized byMS. In other embodiments, as part of the MS sample preparationtechnique, such MS techniques as MALDI can be used to release a compoundfrom the matrix, particularly where a labile bond is used originally totether the compound to the matrix. For instance, a bead selected from alibrary can be irradiated in a MALDI step in order to release thediversomer from the matrix, and ionize the diversomer for MS analysis.

B) Multipin Synthesis

The libraries of the subject method can take the multipin libraryformat. Briefly, Geysen and co-workers (Geysen et al. (1984) PNAS81:3998-4002) introduced a method for generating compound libraries by aparallel synthesis on polyacrylic acid-grated polyethylene pins arrayedin the microtitre plate format. The Geysen technique can be used tosynthesize and screen thousands of compounds per week using the multipinmethod, and the tethered compounds may be reused in many assays.Appropriate linker moieties can also been appended to the pins so thatthe compounds may be cleaved from the supports after synthesis forassessment of purity and further evaluation (c.f., Bray et al. (1990)Tetrahedron Lett 31:5811-5814; Valerio et al. (1991) Anal Biochem197:168-177; Bray et al. (1991) Tetrahedron Lett 32:6163-6166).

C) Divide-Couple-Recombine

In yet another embodiment, a variegated library of compounds can beprovided on a set of beads utilizing the strategy ofdivide-couple-recombine (see, for example, Houghten (1985) PNAS82:5131-5135; and U.S. Pat. Nos. 4,631,211; 5,440,016; 5,480,971).Briefly, as the name implies, at each synthesis step where degeneracy isintroduced into the library, the beads are divided into separate groupsequal to the number of different substituents to be added at aparticular position in the library, the different substituents coupledin separate reactions, and the beads recombined into one pool for thenext iteration.

In one embodiment, the divide-couple-recombine strategy can be carriedout using an analogous approach to the so-called “tea bag” method firstdeveloped by Houghten, where compound synthesis occurs on resin sealedinside porous polypropylene bags (Houghten et al. (1986) PNAS82:5131-5135). Substituents are coupled to the compound-bearing resinsby placing the bags in appropriate reaction solutions, while all commonsteps such as resin washing and deprotection are performedsimultaneously in one reaction vessel. At the end of the synthesis, eachbag contains a single compound.

D) Spatially Addressable Parallel Chemical Synthesis

A scheme of combinatorial synthesis in which the identity of a compoundis given by its locations on a synthesis substrate is termed a spatiallyaddressable synthesis. In one embodiment, the combinatorial process iscarried out by controlling the addition of a chemical reagent tospecific locations on a solid support. For example, a preferred methodfor the combinatorial synthesis of compounds of the invention, forexample analogues of those shown in Tables 1 and 2, is the SPOTtechnology described in EP0651762 with improvements and applicationsdescribed in WO 00/12575, WO 01/18545 and Reinehe et al 2001 (CurrentOpinions in Biotech 12: 59-64). Another preferred method is provided bythe use of microchannels to create combinatorial arrays of candidate orvariant compounds, for example as described in WO 99/67024 and WO99/56878.

Alternatively, combinatorial libraries in a spatially addressable formmay be generated by light-directed synthesis (Dower et al. (1991) AnnuRep Med Chem 26:271-280; Fodor, S. P. A. (1991) Science 251:767; Pirrunget al. (1992) U.S. Pat. No. 5,143,854; Jacobs et al. (1994) TrendsBiotechnol 12:19-26). The spatial resolution of photolithography affordsminiaturization. This technique can be carried out through the useprotection/deprotection reactions with photolabile protecting groups.

The key points of this technology are illustrated in Gallop et al.(1994) J Med Chem 37:1233-1251. A synthesis substrate is prepared forcoupling through the covalent attachment of photolabilenitroveratryloxycarbonyl (NVOC) protected amino linkers or otherphotolabile linkers. Light is used to selectively activate a specifiedregion of the synthesis support for coupling. Removal of the photolabileprotecting groups by light (deprotection) results in activation ofselected areas. After activation, the first of a set of amino acidanalogs, each bearing a photolabile protecting group on the aminoterminus, is exposed to the entire surface. Coupling only occurs inregions that were addressed by light in the preceding step. The reactionis stopped, the plates washed, and the substrate is again illuminatedthrough a second mask, activating a different region for reaction with asecond protected building block. The pattern of masks and the sequenceof reactants define the products and their locations. Since this processutilizes photolithography techniques, the number of compounds that canbe synthesized is limited only by the number of synthesis sites that canbe addressed with appropriate resolution. The position of each compoundis precisely known; hence, its interactions with other molecules can bedirectly assessed.

In a light-directed chemical synthesis, the products depend on thepattern of illumination and on the order of addition of reactants. Byvarying the lithographic patterns, many different sets of test compoundscan be synthesized simultaneously; this characteristic leads to thegeneration of many different masking strategies.

E) Encoded Combinatorial Libraries

In yet another embodiment, the subject method utilizes a compoundlibrary provided with an encoded tagging system. A recent improvement inthe identification of active compounds from combinatorial librariesemploys chemical indexing systems using tags that uniquely encode thereaction steps a given bead has undergone and, by inference, thestructure it carries. Conceptually, this approach mimics phage displaylibraries, where activity derives from expressed peptides, but thestructures of the active peptides are deduced from the correspondinggenomic DNA sequence. The first encoding of synthetic combinatoriallibraries employed DNA as the code. A variety of other forms of encodinghave been reported, including encoding with sequenceable bio-oligomers(e.g., oligonucleotides and peptides), and binary encoding withadditional non-sequenceable tags.

1) Tagging with Sequenceable Bio-Oligomers

The principle of using oligonucleotides to encode combinatorialsynthetic libraries was described in 1992 (Brenner et al. (1992) PNAS89:5381-5383), and an example of such a library appeared the followingyear (Needles et al. (1993) PNAS 90:10700-10704). A combinatoriallibrary of nominally 7⁷ (=823,543) peptides composed of all combinationsof A rg, Gln, Phe, Lys, Val, D-Val and T hr (three-letter amino acidcode), each of which was encoded by a specific dinucleotide (TA, TC, CT,AT, TT, CA and AC, respectively), was prepared by a series ofalternating rounds of peptide and oligonucleotide synthesis on solidsupport. In this work, the amine linking functionality on the bead wasspecifically differentiated toward peptide or oligonucleotide synthesisby simultaneously preincubating the beads with reagents that generateprotected OH groups for oligonucleotide synthesis and protected NH₂groups for peptide synthesis (here, in a ratio of 1:20). When complete,the tags each consisted of 69-mers, 14 units of which carried the code.The bead-bound library was incubated with a fluorescently labeledantibody, and beads containing bound antibody that fluoresced stronglywere harvested by fluorescence-activated cell sorting (FACS). The DNAtags were amplified by PCR and sequenced, and the predicted peptideswere synthesized. Following such techniques, compound libraries can bederived for use in the subject method, where the oligonucleotidesequence of the tag identifies the sequential combinatorial reactionsthat a particular bead underwent, and therefore provides the identity ofthe compound on the bead.

The use of oligonucleotide tags permits exquisitely sensitive taganalysis. Even so, the method requires careful choice of orthogonal setsof protecting groups required for alternating co-synthesis of the tagand the library member. Furthermore, the chemical lability of the tag,particularly the phosphate and sugar anomeric linkages, may limit thechoice of reagents and conditions that can be employed for the synthesisof non-oligomeric libraries. In preferred embodiments, the librariesemploy linkers permitting selective detachment of the test compoundlibrary member for assay.

Peptides have also been employed as tagging molecules for combinatoriallibraries. Two exemplary approaches are described in the art, both ofwhich employ branched linkers to solid phase upon which coding andligand strands are alternately elaborated. In the first approach (Kerret al. (1993) JACS 115:2529-2531), orthogonality in synthesis isachieved by employing acid-labile protection for the coding strand andbase-labile protection for the compound strand.

In an alternative approach (Nikolaiev et al. (1993) Pept Res 6:161-170),branched linkers are employed so that the coding unit and the testcompound can both be attached to the same functional group on the resin.In one embodiment, a cleavable linker can be placed between the branchpoint and the bead so that cleavage releases a molecule containing bothcode and the compound (Ptek et al. (1991) Tetrahedron Lett32:3891-3894). In another embodiment, the cleavable linker can be placedso that the test compound can be selectively separated from the bead,leaving the code behind. This last construct is particularly valuablebecause it permits screening of the test compound without potentialinterference of the coding groups. Examples in the art of independentcleavage and sequencing of peptide library members and theircorresponding tags has confirmed that the tags can accurately predictthe peptide structure.

2) Non-Sequenceable Tagging: Binary Encoding

An alternative form of encoding the test compound library employs a setof non-sequencable electrophoric tagging molecules that are used as abinary code (Ohlmeyer et al. (1993) PNAS 90:10922-10926). Exemplary tagsare haloaromatic alkyl ethers that are detectable as theirtrimethylsilyl ethers at less than femtomolar levels by electron capturegas chromatography (ECGC). Variations in the length of the alkyl chain,as well as the nature and position of the aromatic halide substituents,permit the synthesis of at least 40 such tags, which in principle canencode 2⁴⁰ (e.g., upwards of 10¹²) different molecules. In the originalreport (Ohlmeyer et al., supra) the tags were bound to about 1% of theavailable amine groups of a peptide library via a photocleavableo-nitrobenzyl linker. This approach is convenient when preparingcombinatorial libraries of peptide-like or other amine-containingmolecules. A more versatile system has, however, been developed thatpermits encoding of essentially any combinatorial library. Here, thecompound would be attached to the solid support via the photocleavablelinker and the tag is attached through a catechol ether linker viacarbene insertion into the bead matrix (Nestler et al. (1994) J Org Chem59:4723-4724). This orthogonal attachment strategy permits the selectivedetachment of library members for assay in solution and subsequentdecoding by ECGC after oxidative detachment of the tag sets.

Although several a mide-linked libraries in the art employ binaryencoding with the electrophoric tags attached to amine groups, attachingthese tags directly to the bead matrix provides far greater versatilityin the structures that can be prepared in encoded combinatoriallibraries. Attached in this way, the tags and their linker are nearly asunreactive as the bead matrix itself. Two binary-encoded combinatoriallibraries have been reported where the electrophoric tags are attacheddirectly to the solid phase (Ohlmeyer et al. (1995) PNAS 92:6027-6031)and provide guidance for generating the subject compound library. Bothlibraries were constructed using an orthogonal attachment strategy inwhich the library member was linked to the solid support by aphotolabile linker and the tags were attached through a linker cleavableonly by vigorous oxidation. Because the library members can berepetitively partially photoeluted from the solid support, librarymembers can be utilized in multiple assays. Successive photoelution alsopermits a very high throughput iterative screening strategy: First,multiple beads are placed in 96-well microtiter plates; second,compounds are partially detached and transferred to assay plates; third,a metal binding assay identifies the active wells; fourth, thecorresponding beads are rearrayed singly into new microtiter plates;fifth, single active compounds are identified; and sixth, the structuresare decoded.

The peptidomimetic compounds of the present invention may be synthesizedusing techniques such as those described above to provide a large,highly diverse library of candidate inhibitors, because compounds of theinvention can be readily prepared by successively forming a series ofcarbon-heteroatom bonds, such as amide or urea bonds, under mildconditions. Thus, from a discrete set of subunits, such as amino acidsand subunits which incorporate a bicyclic aryl-1,2diazacyclohexanesubunit, a wide range of combinations and permutations of these subunitsmay be rapidly and easily synthesized and tested for biologicalactivity.

EXEMPLIFICATION

The present invention will now be illustrated by reference to thefollowing examples, which set forth particularly advantageousembodiments. However, it should be noted that these embodiments areillustrative and are not to be construed as restricting the invention inany way.

Example 1 Preparation of Peptidomimetic Compounds

Peptidomimetic compounds were prepared by assembly of building blocksusing standard solid phase peptide chemistry (R. B. Merrifield, J. Am.Chem. Soc. 85, 2149-2154 (1963), G. Barany, R. B. Merrifield in ThePeptides, Vol. 2 (eds. E. Gross, J. Meienhofer) 1-284 (Academic, NewYork; 1980)) in a peptide synthesizer (ACT90, Advanced ChemTech) andpurified by high performance liquid chromatography (HPLC).

HPLC was conducted on a Vision Chromatograph (PerSeptive Biosystems).Analytical HPLC was performed in reverse phase mode using watersμBondapak C₁₈ columns (0.46×25 cm, 5μ or 0.39×30 cm, 10 μl) or NucleosilC₁₈ columns (0.46×25 cm, 5μ or 0.4×30 cm, 10μ) from CS-ChromatographieService, Langerwehe, Germany. Preparative HPLC was performed in reversedphase mode using Waters μBondapak C₁₈ columns (1.9×30 cm, 10μ) orNucleosil C₁₈ columns (2.0×30 cm, 10μ) from CS-Chromatographie Service.Flash chromatography was performed on Merck Kieselgel 60 (0.063-0.200mm, Art No. 1.07734) obtained from Merck Darmstadt, Germany. T. L. C.was performed on aluminium sheets Silica gel 60 F₂₅₄ (Art No. 1.05554)obtained from Merck Darmstadt, Germany. ¹H-NMR-Spectra were determinedat 200 MHz using tetramethylsilane as internal standard, and areexpressed as chemical shift (δ) values in parts per million relative totetramethylsilane and assigned using s=singlet; m=multiplet; d=doublet;t=triplet; q=quartet, sp=septet, br=broad.

The following abbreviations are used: Boc=tert-butoxycarbonyl,Fmoc=9-fluorenylmethoxycarbonyl, Acm=acetamidomethyl,Prm=propylamidomethyl, DCM=dichloromethane; DMF=N,N-dimethylformamide,DMAP=4-dimethylamino pyridine, HOBt=1-hydroxybenzotriazole;DIC=diisopropylcarbodiimide,TBTU=2-(1-H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate, THF=tetrahydrofuran, DIPEA=diisopropylethylamine,TFA=trifluoroacetic acid, Me=methyl, Ac=acetyl, tBu=tert-butyl,Bn=benzyl, Ph=phenyl, h=hour(s), min=minute(s), aq.=aqueous, r.t.=roomtemperature (18-26° C.), Pmc=2,2,5,7,8-pentamethylchromane-6-sulfonyl,PyBOP=benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate, Z=benzyloxycarbonyl,EDCI=1-ethyl-3(3′-dimethylaminopropyl)carbodiimide,DBU=1,8-diazabicyclo[5.4.0]undec-7-ene, Cit=(L) citrullinyl,Cha=(L)-cyclohexylalaninyl, Gpg=(L)-N-amidino-4-piperidinylglycinyl,βPhPro=2-(S)-3-(R)-3-phenylprolinyl,Tic=(L)-tetrahydroisoquinoline-3-carbonyl,azaTic=3,4-dihydro-1H-phthalazine-2-carbonyl, Disc=(D,L)1,2-dihydro-2H-isoindole carbonyl,Thiq=(L)-tetrahydroisoquinoline-1-carbonyl, Hbc(D,L)-2,3,4,5-tetrahydro-1H-benzo[d]azepine-2-carbonyl, Haic=(2S,5S)-5-amino-1,2,3,4,5,6,7-hexahydro-azepino [3,2,1-h,i]indole-4-one-2-carbonyl, Odapdc=(1S,9S)-9-aminooctahydro-6,10-dioxo-6H-pyridazino-[1,2-a][1,2]diazepine-1-carbonyl,[SΨ(oxaz)L]=oxazole mimetic of S-L, [SΨ(imid)L]=imidazole mimetic ofS-L, A=Ala=(L)-alaninyl, R=Arg=(L)-argininyl, C=Cys=(L)-cysteinyl,F=Phe=(L)-phenylalaninyl, V=Val=(L)-valinyl, Met=(L)-methioninyl,Nle=(L)-norleucinyl, S=Ser=(L)-serinyl, L=Leu=(L)-leucinyl,alle=(L)-alloisoleucinyl, Nva=(L)-norvalinyl, Pya=(L)-pyridylalaninyl,Om=(L)-omithinyl, Chg=(L)-cyclohexylglycinyl,Hfe=(L)-homophenylalaninyl, Thi=(L)-2-thienylalaninyl,Coa=(L)-cyclooctylalaninyl, Nba=(L)-norbornylalaninyl,

N-(2-phenylethyl)ethanolamine was prepared from 2-phenylethyl chlorideand ethanolamine according to literature procedure (J. Barbiere, Bull.Soc. Chim. Fr. 5, 7, 1940, 621). Commercially available Fmoc aminoacids, HOBt, TBTU and PyBOP were purchased from Advanced ChemTech,Novabiochem, Bachem, Neosystems or RSP Amino Acid Analogues. All otherchemicals and solvents were purchased from Merck Darmstadt orSigma-Aldrich-Fluka and used without further purification. DMF was driedover molecular sieves 4 Å for at least 4 weeks, stirred over acidicaluminium oxide for 20 minutes to remove traces of amines, and filteredthrough a 0.2 μm filter prior use.

Table 1 (a, b and c) lists certain compounds according to Formulae I andII that are exemplary of the invention. Table 2 lists other compoundsexamined for their immunomodulatory and other properties using theassays described herein. As will be apparent to a person skilled in theart after reading this disclosure, peptidomimetics shorter than theheptamers set forth in Table 1 are useful for certain applications. Assuch, shorter peptidomimetics also form part of this invention.Preferred lengths of these shorfter peptides are tetra- or pentamers.

Example 2 Preparation of Ac-Cha-Gpg-Tic-Nle-βPhPro-[SΨ(oxaz)L]NMe₂ (P53)2.1 Preparation of Fmoc-βPhPro-OH

44.61 g N-Acetyl-trans-3(R)-phenyl-(S)-proline-1-(S)-phenylethyl amide(J. Y. L. Chung, J. T. Wasicak, W. A. Arnold, C. S. May, A. N. Nadzan,M. W. Holladay, J. Org. Chem. 1990, 55, 270-275) was dissolved in 730 ml8 N HCl and 360 ml acetic acid. The resulting solution was heated to140° C. for 16 h. After cooling to r.t. the solution was evaporated todryness. The residue was taken up in 1000 ml of water. The aq. solutionwas washed with ethyl acetate (3×200 ml) and concentrated under reducedpressure to a final volume of 300 ml. 400 ml aq. 10% Na₂CO₃-solutionwere added and the aq. layer was washed with ethyl acetate (4×200 ml).200 ml aq. 10% Na₂CO₃-solution were added and the solution was cooled to0° C. A solution of 51.22 g FmocCl in 300 ml dioxane was added dropwiseover 1.5 h and the resulting suspension was stirred at r.t. for 18 h.The precipitate was removed by decantation. The aq. solution was washedwith diethyl ether (1×200 ml) acidified with 1 N HCl to pH 3 andextracted with D CM (2×300 ml). The precipitate was dissolved in ethylacetate. The resulting solution was extracted with saturated aq. NaHCO₃solution. The aq. layer was acidified with conc. HCl to pH 3 andextracted with DCM. The combined DCM layers were dried over Na₂SO₄,filtered and evaporated to dryness. The residue was preabsorbed ontosilica and purified by flash chromatography using ethylacetate/hexane/acetic acid 150:50:1 as eluent. The fractions containingthe desired product (checked by T. L. C.) were combined and evaporated.The resulting residue was recrystallized from CHCl₃/hexane 1:2 to give atotal yield of 24.92 g (55%).

¹H-NMR (CDCl₃): 1.95-2.15 (m, 1H, FmocNCH₂CHH, 2.24-2.47 (m, 1H,FmocNCH₂CHH), 3.43-3.85 (m, 3H), 4.06-4.58 (m, 4H), 6.2 (br s, 1H, COOH)7.11-7.82 (m, 13H, arom. Hs).

2.2 Preparation of H[S(OtBu)Ψ(oxaz)L]NMe₂ 2.2.1 Preparation of DipeptideI:

10.0 g Methyl N-(diphenylmethylen)glycinate (M. J. O'Donnell, R. L.Polt, J. Org. Chem. 1982, 47, 2663-2666) were added to a solution of4.87 g KOtBu in 100 ml dry THF at −5° C. and the resulting solution wasstirred at 0° C. for 15 min. This solution was added over 3.5 h to asolution of 7.1 ml isobutyric acid chloride in 300 ml dry THF at −78° C.After addition was completed the orange reaction mixture was allowed toreach r.t. The resulting yellow solution was treated with 200 ml 1 N HCland the mixture was stirred at r.t. for 15 min. The organic solvent wasevaporated under reduced pressure. The aq. layer was washed with ethylacetate (4×100 ml) and evaporated to dryness to give 10.2 g residue.

11.66 g Z-Ser(tBu)OH was dissolved in 200 ml dry THF under an atmosphereof Ar and the solution was cooled to −18° C. 5.48 ml Triethylamine wereadded followed by 5.2 ml isobutyl chloroformate. The suspension wasstirred at −18° C. for 15 min. The above residue was added and asolution of 5.48 ml triethylamine in 80 ml dry THF was added dropwiseover 1 h. After addition was completed the suspension was stirred at−18° C. for additional 1.5 h and then allowed to reach r.t. Saturatedaq. NaCl solution (200 ml) was added and the mixture was stirred for 15min. The aq. layer was separated and extracted with diethyl ether (3×100ml). The combined organic layers were washed with pH 7 phosphate buffer(1×50 ml), dried over Na₂SO₄, filtered and evaporated to dryness. Theresidue was purified by flash chromatography using ethyl acetate/hexane(1:3-1:2) as eluent to give 14.4 g (84%) of the desired compound as amixture of diastereomers. ¹H-NMR (CDCl₃): 1.09-1.28 (m 15H, CH{umlautover (Me)}₂, tBu), 3.05 (sp, 1H, CHMe₂), 3.38-3.45 (m, 1H, CHHOtBu),3.78, 3.79 (2s, 3H, OMe), 3.75-3.88 (br s, 1H, CHHOtBu), 5.05-5.18 (m,2H, CH ₂Ph), 5.38 (d, J=6.8 Hz, 1H, COCHCO), 5.70 (br s, 1H,carbamate-NH), 7.25-7.42 (m, 5H, Ph), 7.95 (br s, 1H, amide-NH).

2.2.2 Preparation of Z[S(OtBu)Ψ(oxaz)L]OMe

55.0 g Triphenylphosphineoxide and 31.3 ml DBU were added to a solutionof 30.5 g of the dipeptide I in 38 ml dry carbontetrachloride, 38 ml dryacetonitrile and 38 ml dry pyridine at 0° C. The resulting mixture wasstirred at r.t. for 20 h. The solvents were removed under reducedpressure and the residue was coevaporated with toluene (4×150 ml). Theresulting residue was dissolved in 900 ml DCM. The solution was washedwith aq. 5% KHSO₄-solution (5×200 ml) and pH 7 phosphate buffer (2×150ml), dried over Na₂SO₄, filtered and evaporated to dryness. The residuewas taken up in 450 ml ethyl acetate and the suspension was sonicatedand filtered. The filtrate was concentrated to a final volume of 100 ml.300 ml hexane were added and the resulting suspension was againsonicated and filtered. The filtrate was evaporated to dryness and theresidue was purified by flash chromatography using ethyl acetate/hexane(1:3) as eluent to give 24.7 g (85%) of the title compound as a slightlyyellow oil. ¹H-NMR (CDCl₃): 1.07 (s, 9H, tBu), 1.23-1.28 (m, 6H, CHMe_(2),) 3.64 (dd, J3=, 1H, 4.0, 9.2 Hz, 1H, CHHOtBu), 3.68-3.85 (m, 2H,CHMe ² , CHHOtBu), 3.90 (s, 3H, OMe), 5.00-5.11 (br m, 1H, CHNHCO), 5.13(s, 2H, CH₂Ph), 5.79 (br d, J=7.4 Hz, 1H, NH), 7.25-7.41 (m, 5H, Ph).

2.2.3 Preparation of Z[S(OtBu)Ψ(oxaz)L]NMe₂

24.71 g Z[S(OtBu)Ψ(oxaz)L]OMe was dissolved in 200 ml methanol and thesolution was cooled to 0° C. A solution of 1.84 g LiOH in 80 ml waterwas added dropwise over 35 min. The mixture was stirred at 0° C. for 1.5h and at r.t. for 16 h. The solution was neutralized with 1 N HCl andmethanol was evaporated under reduced pressure. The resulting aq.solution was acidified to pH 4 with 1 N HCl and extracted with D CM(4×100 ml). The combined organic layers were dried over Na₂SO₄, filteredand evaporated. The residue was dissolved in 250 ml dry DMF and thesolution was cooled to 0° C. After addition of 11.3 g HOBt, 14.1 g EDCI,7.6 ml triethylamine, 13.8 g dimethylamine hydrochloride and another15.7 ml triethylamine the mixture was stirred for 17 h at r.t. Thesolution was evaporated under reduced pressure and the resulting residuewas coevaporated with toluene (1×100 ml). The residue was taken up in400 ml ethyl acetate, the resulting suspension was filtered and thefiltrate was washed with aq. 5% KHSO₄ solution (3×100 ml), aq. saturatedNaHCO₃ solution (2×100 ml) and pH 7 phosphate buffer (2×100 ml). Theorganic layer was dried over Na₂SO₄, filtered and evaporated. Theresidue was purified by flash chromatography using ethyl acetate/hexane(2:3) as eluent to give 23.3 g (92%) of the title compound as an oil.¹H-NMR (CDCl₃): 1.07 (s, 9H, tBu), 1.22-1.26 (m, 6H, CHMe ₂), 3.03, 3.18(2 s, 2×3H, NMe₂), 3.51 (sp, J3=7.0 Hz, 1H, CHMe₂), 3.65 (dd, J=4.0, 8.8Hz, 1H, CHHOtBu), 3.78 (br m, 1H. CHHOtBu), 4.98-5.09 (br m, 1H,CHNHCO), 5.10-5.21 (m, 2H, CH₂Ph), 5.70 (br d, J=7.3 Hz, 1H, NH),7.21-7.45 (m, 5H, Ph).

2.2.4 Preparation of H[S(OtBu)Ψ(oxaz)L]NMe₂

A solution of 23.3 g Z[S(OtBu)Ψ(oxaz)L]NMe₂ in 100 ml ethanol was addedto a suspension of 2.35 g Pd/C (10%) under an atmosphere of hydrogen andthe mixture was stirred at r.t. for 18 h. The suspension was filteredthrough Celite and the filtrate was evaporated to dryness to give 14.5 g(90%) of the title compound. ¹H-NMR (CDCl₃): 1.15 (s, 9H, tBu), 1.27 (d,J=7.0 Hz, 6H, CHMe ₂), 2.04 (s, 2H, NH₂), 3.04, 3.23 (2 s, 2×3H, NMe₂),3.50 (sp, J=7.0 Hz, 1H, CHMe ² ), 3.60 (dd, J=6.5, 8.8 Hz, 1H, CHHOtBu),3.69 (dd, J=4.4, 8.8 Hz, 1H, CHHOtBu), 4.14 (dd, J=4.4, 6.5 Hz, 1H,CHNH₂).

2.3 Preparation of HβPhPro-2-chlorotrityl Resin

A solution of 7.4 g Fmoc-βPhPro-OH in 120 ml dry DCM was added to 12.0 g2-chlorotrityl chloride resin (0.83 mmol/g, Novabiochem). DIPEA (3.0 ml)was added and the mixture was shaken for 10 min. Additional 4.5 ml DIPEAwere added and shaking was continued for 145 min. Methanol (10 ml) wasadded the mixture was shaken for another 25 min. The resin was filteredoff, washed with DCM (5×100 ml), methanol (2×100 ml) and DCM (4×100 ml).A small sample was dried carefully and deprotected with DCM/piperidine(1:1) for 30 min. Photometric determination of the resultingFmoc-piperidine adduct (absorption at 301 nm) gave a resin loading of0.54 mmol/g. The remaining resin was treated with 100 ml DCM and 80 mlpiperidine at r.t. for 160 min, washed with DCM (10×100 ml) and diethylether (4×80 ml) and dried in vacuo to give 14.37 gHβPhPro-2-chlorotrityl resin.

2.4. Preparation of Ac-Cha-Gpg-Tic-Nle-βPhPro-[SΨ(oxaz)L]NMe₂ (P53)

The peptididomimetic was prepared by Fmoc solid phase synthesis startingwith HβPhPro-2-chlorotrityl chloride resin (2416 mg, 1.3 mmol) in a 50ml reaction vessel fitted with a frit in the bottom (Advanced ChemTechACT90).

Resin swelling was carried out by treating the resin with DMF (4×1min.). The resin was deprotected using a 20% solution of piperidine inDMF (1×3 min, 1×7 min, 20 ml each) and subsequently washed with DMF(10×20 ml). Acylation was carried out by addition of FmocNleOH (1380 mg,3.9 mmol), DMF (8.2 ml), HOBt (600 mg, 3.9 mmol), and DIC (0.61 ml, 3.9mmol). The coupling was left for 18 h, washed with DMF (7×20 ml). Asmall portion was checked for completion of acylation using theChloranil test (J. Blake, C. H. Li, Int. J. Peptide Protein Res., 1975,7, 495). The resin was capped using a solution of acetic anhydride (2 M)and DMAP (0.1 M) in DMF (20 ml, 1×10 min) and subsequently washed withDMF (12×20 ml). The resin was deprotected, washed, capped and washed asabove and coupled with FmocTicOH (1.56 g, 3.9 mmol), TBTU (1.26 g, 3.9mmol) and DIPEA (0.71 ml, 4.16 mmol) in 8 ml DMF for 75 min.

The resin was deprotected, washed, capped and washed as above andcoupled with FmocGpg(Pmc)OH (1.35 g, 1.95 mmol), HOBt (0.3 g, 1.95 mmol)and DIC (0.305 ml, 1.95 mmol) in 7 ml DMF for 16 h.

The resin was deprotected, washed, capped and washed as above andcoupled with FmocChaOH (1.54 g, 3.9 mmol), HOBt (0.6 g, 3.9 mmol) andDIC (0.61 ml, 3.9 mmol) in 8 ml DMF for 3 h.

Deprotection and washing was carried out as above and capping wasperformed by treatment with acetic anhydride (2 M) and DMAP (0.1 M) in20 ml DMF for 3×20 min. The resin was washed with DMF (12×20 ml), MeOH(3×50 ml), Et₂O (3×40 ml) and dried in vacuo.

The resin was treated with 33 ml DCM/trifluoroethanol/acetic acid(8:1:1) at r.t. for 45 min, filtered and washed with 65 mlDCM/trifluoroethanol/acetic acid (8:1:1) and 100 ml DCM. 250 ml n-Hexanewere added to the filtrate, the resulting suspension was evaporated todryness and the residue was coevaporated with n-hexane (3×100 ml) togive 1.188 g of crude Ac-Cha-Gpg(Pmc)-Tic-Nle-βPhPro-OH. The residue wasdissolved in 5 ml dry DMF and cooled to 0° C. After addition of 332 mgHOBt, 642 mg H[S(OtBu)Ψ(oxaz)L]NMe₂ and 415 mg EDCI, the solution wasstirred for 1.5 h at 0° C. and another 16 h at r.t. The solvent wasremoved under reduced pressure and the residue was dissolved in 80 mlethyl acetate. The solution was washed with aq. 5% KHSO₄ solution (1×40ml), saturated aq. NaHCO₃ solution and pH 7 phosphate buffer, dried overNa₂SO₄, filtered and evaporated to dryness. The residue was treated with20 ml of TFA/water/thioanisole/1,2-ethanedithiol/triethylsilane85.5:5:5:2.5:2 for 3.7 h at r.t. The product was precipitated by adding550 ml of chilled diethyl ether to the solution. The suspension wascentrifuged at 3300 rpm for 10 min, the supernatant was discarded, theprecipitate was resuspended in chilled ether, centrifuged again and thesupernatant was once again discarded. The precipitate was dissolved inacetonitrile and 0.1% aq TFA. The organic solvents were evaporated underreduced pressure and the aq. solution was freeze dried. The crudeproduct (931 mg) was purified by HPLC using a gradient of acetonitrilein 0.1% aq TFA to yield 584 mg pureAc-Cha-Gpg-Tic-Nle-βPhPro-[SΨ(oxaz)L]NMe₂×TFA. The product wascharacterized by mass spectrometry, MALDI-TOF MS: M/Z=1064.57, (MH+),1102.53 (MK+).

Example 3 Preparation of Ac-Cha-Arg-Tic-Nle-βPhPro-[SΨ(oxaz)L]NMe₂ (P51)

This peptiodomimetic was prepared using similar methodology to thatdescribed for Example 2.4 but on a smaller scale using less resin, a 25ml reaction vessel and 7 ml solvent portions for swelling, washing,capping, deprotection and coupling during solid phase synthesis.Coupling on βPhPro was performed using FmocNleOH, HOBt, DIC (3equivalents each) for 16 h, coupling on Nle was done using FmocTicOH(3eq), TBTU (3 eq) and DIPEA (3.2 eq) for 1.5 h, coupling on Tic wasdone using FmocArg(Pmc)OH, HOBt, DIC (3 eq each) for 16 h, coupling onArg was done using FmocChaOH, HOBt, DIC (3 eq each) for 3 h. Thesolution coupling was carried out using H[S(OtBu)Ψ(oxaz)L]NMe₂ (2 eq),PyBOP (2 eq) and 4-methylmorpholine (4 eq) in DMF at 0° C. for 1 h andat r.t. for 16 h. The resulting mixture was evaporated to dryness andthe residue was treated withTFA/water/thioanisole/1,2-ethanedithiol/triethylsilane 85.5:5:5:2.5:2for 3.5 h at r.t. The product was precipitated by adding 200 ml ofchilled diethyl ether. The suspension was kept at 0° C. for 1 h and thantreated as described in example 2.4. The crude product was purified byHPLC using a gradient of acetonitrile in 0.1% aq TFA. MALDI-TOF MS:M/Z=1038.70 (MH+), 1060.36 (MNa+), 1076.61 (MK+).

Example 4 Preparation of Ac-Cha-Arg-Tic-Met-βPhPro-[SΨ(oxaz)L]NMe₂ (P33)& Ac-Cha-Gpg-Tic-Met-βPhPro-[SΨ(oxaz)L]NMe₂ (P60)

Peptiodomimetic P33 (Arg) was prepared as described in Example 3, exceptthat the resin was coupled in coupling step 1 with FmocMetOH instead ofFmocNleOH. MALDI-TOF MS: M/Z=1057.00 (MH+), 1079.01 (MNa+), 1094.98(MK+).

Peptiodomimetic P60 (Gpg) was prepared as described in Example 3, exceptthat the resin was coupled in coupling step 1 with FmocMetOH instead ofFmocNleOH, and in coupling step 3 with FmocGpg(Pmc)OH, HOBt, DIC (1.5 eqeach) instead of FmocArg(Pmc)OH, HOBt and DIC (3 eq each). MALDI-TOF MS:M/Z=1082.54 (MH+), 1120.51 (MK+).

Example 5 Preparation of Ac-Cha-Arg-Tic-Met(O)-βPhPro-[SΨ(oxaz)L]NMe₂(P43) & Ac-Cha-Gpg-Tic-Met(O)-βPhPro-[Sψ(oxaz)L]NMe₂ (P47)

Peptiodomimetic P43 (Arg) was prepared as described in Example 3, exceptthat the resin was coupled in coupling step 1 with FmocMet(O)OH insteadof FmocNleOH. MALDI-TOF MS: M/Z=1072.57 (MH+).

Peptiodomimetic P47 (Gpg) was prepared as described in Example 3, exceptthat the resin was coupled in coupling step 1 with FmocMet(O)OH insteadof FmocNleOH, and in coupling step 3 with FmocGpg(Pmc)OH, HOBt, DIC (1.5eq each) instead of FmocArg(Pmc)OH, HOBt, DIC (3 eq each). MALDI-TOF MS:M(Z=1098.73 (MH+), 1120.70 (MNa+), 1136.68 (MK+).

Example 6 Preparation of Ac-Cha-Arg-Disc-Met-βPhPro-[SΨ(oxaz)L]NMe₂(P40) & Ac-Cha-Gpg-Disc-Met-βPhPro-[SΨ(oxaz)L]NMe₂ (P41)

Peptiodomimetic P40 (Arg) was made as P33 in Example 4 except for thatthe resin was coupled in coupling step 2 with racemic FmocDiscOH insteadof FmocTicOH. The resulting two diastereomers were separated at thefinal HPLC step using a gradient of acetonitrile in 0.1% aq TFA. Eachstereoisomer was isolated and denoted as either the “fast” fraction(suffixed-1 in the compound names) or the “slow” fraction (suffixed-2 inthe compound names), and tested separately in the subsequent biologicalassays. MALDI-TOF MS (P40-1): M/Z=1042.67 (MH+), 1058.66 (MNa+), 1080.63(MK+). MALDI-TOF MS (P40-2): M/Z=1042.69 (MH+), 1058.66 (MNa+), 1080.62(MK+).

Peptiodomimetic P41 (Gpg) was prepared as P60 in Example 4 except forthat the resin was coupled in coupling step 2 with racemic FmocDiscOHinstead of FmocTicOH. The resulting two diastereomers were separated atthe final HPLC step using a gradient of acetonitrile in 0.1% aq TFA.Each stereoisomer was isolated and denoted as either the “fast” fraction(suffixed-1 in the compound names) or the “slow” fraction (suffixed-2 inthe compound names), and tested separately in the subsequent biologicalassays.

MALDI-TaOF MS (P41-1): M/Z=1068.43 (MH+), 1106.38 (MK+). MALDI-TOF MS(P41-2): M/Z=1068.42 (MH+), 1106.36 (MK+).

Example 7 Preparation of Ac-Cha-Gpg-Tic-Nle-NHCH₂CH₂Ph (P69) &Ac-Cha-Arg-Tic-Nle-NHCH₂CH₂Ph (P82) 7.1 Preparation ofHNle-2-chlorotrityl Resin

The resin was prepared using similar methodology to that described inExample 2 step 2.3 from FmocNleOH (4.37 g) and 2-chlorotrityl chlorideresin (7.45 g, 0.83 mmol/g, Novabiochem) to yield 7.77 gHNle-2-chlorotrityl resin (loading: 0.50 mmol/g).

7.2 Preparation of Ac-Cha-Gpg(Pmc)-Tic-Nle-OH

The peptidomimetics were prepared using similar methodology to thatdescribed for Example 1.4 by deprotection and coupling usingHNle-2-chlorotrityl resin (2.52 g). Coupling on Nle was performed usingTBTU (1.09 g, 1.35 mmol), DIPEA (0.62 ml, 1.44 mmol) and FmocTicOH (1.36g, 1.35 mmol) in 7 ml DMF with a coupling time of 2.5 h. Coupling on Ticwas carried out using FmocGpg(Pmc)OH (1.17 g, 0.68 mmol), HOBt (0.26 g,0.68 mmol) and DIC (0.27 ml, 0.68 mmol) in 6 ml DMF over 17 h andcoupling on Gpg was done with FmocChaOH (1.34 g, 1.35 mmol), HOBt (0.52g, 1.35 mmol) and DIC (0.53 ml, 1.35 mmol) in 6 ml DMF over 2.5 h.Completion of coupling was checked by Kaiser test or Chloroanil test,respectively (E. Kaiser, et al. (1970) Anal. Biochem. 34, 595; J. Blake,C. H. Li, Int. J. Peptide Protein Res., 1975, 7, 495). After finalacetylation, resin cleavage and evaporation (analog to example 1.4) 1.59g crude Ac-Cha-Gpg(Pmc)-Tic-Nle-OH was isolated.

7.3 Preparation of Ac-Cha-Gpg-Tic-Nle-NHCH₂CH₂Ph (P69)

A solution of Ac-Cha-Gpg(Pmc)-Tic-Nle-OH (730 mg, 0.78 mmol) in 5 ml dryDMF was treated with HOBt (239 mg, 1.56 mmol), PyBOP (812 mg, 1.56 mmol)and 2-Phenylethylamine (0.45 ml, 3.65 mmol) at 0° C. The reaction wasstirred at 0° C. for 1.5 h and at r.t. for 13 h. The solvent wasevaporated under reduced pressure and the residue was treated with 10 mlof TFA/water/thioanisole/1,2-ethanedithiol/triethylsilane 85.5:5:5:2.5:2for 4 h at r.t. The product was precipitated by adding 500 ml of chilleddiethyl ether to the solution. The suspension was centrifuged at 3300rpm for 10 min, the supernatant was discarded, the precipitate wasresuspended in chilled ether, centrifuged again and the supernatant wasonce again discarded. The precipitate was dissolved in acetonitrile and0.1% aq TFA. The organic solvents were evaporated under reduced pressureand the aq. solution was freeze dried. The crude product (767 mg) waspurified by HPLC using a gradient of acetonitrile in 0.1% aq. TFA toyield 256 mg pure Ac-Cha-Gpg-Tic-Nle-NHCH₂CH₂Ph. The product wascharacterized by mass spectrometry, MALDI-TOF MS:M/Z=771.310, (MH+),793.286 (MNa+), 809.257 (MK+).

Peptiodomimetic P82 was prepared using similar methodology to thatdescribed for Example 3 but using HNle-2-chlorotrityl resin (Example7.1) instead of HβPhPro-2-chlorotrityl chloride resin. Coupling on Nlewas done using FmocTicOH (3 eq), TBTU (3 eq) and DIPEA (3.2 eq) for 1.5h, coupling on Tic was done using FmocArg(Pmc)OH, HOBt, DIC (3 eq each)for 14 h, coupling on Arg was done using FmocChaOH, HOBt, DIC (3 eqeach) for 3 h. The solution coupling was carried out usingN-(2-Phenylethyl)amine (4 eq), HOBt (2 eq) and PyBOP (2 eq) in DMF at 0°C. for 1 h and at r.t. for 15 h. The resulting mixture was treated asdescribed in Example 3. The crude product was purified by HPLC using agradient of acetonitrile in 0.1% aq TFA. MALDI-TOF MS: M/Z=745.61 (MH+),767.56 (MNa+).

Example 8 Preparation of Ac-Cha-Arg-Tic-Nle-N(Me)Bn (P71) &Ac-Cha-Gpg-Tic-Nle-N(Me)Bin (P74)

Peptiodomimetic P71 (Gpg) was prepared as P82 in Example 7 except forthat crude Ac-Cha-Arg(Pmc)-Tic-Nle-OH was reacted withN-methylbenzylamine (4 eq) instead of N-(2-phenylethyl)amine. HPLC usinga gradient of acetonitrile in 0.1% aq TFA yielded the desired product.MALDI-TOF MS:M/Z=745.56 (MH+), 783.49 (MK+).

Peptiodomimetic P74 (Gpg) was prepared as P69 in Example 7 except forthat crude Ac-Cha-Gpg(Pmc)-Tic-Nle-OH was reacted withN-methylbenzylamine (4 eq) instead of 2-phenylethylamine. HPLC using agradient of acetonitrile in 0.1% aq TFA yielded the desired product.MALDI-TOF MS (P41-1): M/Z=771.63 (MH+), 793.63 (MNa+).

Example 9 Preparation of Ac-Cha-Arg-Disc-Nle-N(Me)Bn (P72) &Ac-Cha-Gpg-Disc-Nle-N(Me)Bn (P76)

Peptiodomimetic P72 was prepared using similar methodology to thatdescribed for P71 in Example 8, except for using racemic FmocDiscOHinstead of FmocTicOH in coupling step 1. The resulting two diastereomerswere separated at the final HPLC step using a gradient of acetonitrilein 0.1% aq TFA and denoted as described in Example 6. MALDI-TOF MS(P72-1): M/Z=731.57 (MH+), 753.55 (MNa+), 769.52 (MK+). MALDI-TOF MS(P72-2): M/Z=731.56 (MH+), 753.55 (MNa+), 769.52 (MK+).

Peptiodomimetic P76 was prepared using similar methodology to thatdescribed for P72 in Example 9, except for using FmocGpg(Pmc)OH, HOBt,DIC (1.5 eq each) instead of FmocArg(Pmc)OH, HOBt, DIC (3 eq each) incoupling step 2. The resulting two diastereomers were separated at thefinal HPLC step using a gradient of acetonitrile in 0.1% aq TFA anddenoted as described in Example 6.1. MALDI-TOF MS (P76-1): M/Z=757.31(MH+), 779.27 (MNa+), 795.24 (MK+). MALDI-TOF MS (P76-2): M/Z=757.37(MH+), 779.34 (MNa+), 795.31 (MK+).

Example 10 Preparation of Ac-Cha-Arg-Tic-Met-NH₂ (P1) &Ac-Cha-Gpg-Tic-Met-NA₂ (P67) 10.1 Preparation of FmocMet-RinkamideResin:

FmocMet-Rinkamide resin was prepared by Fmoc solid phase synthesisstarting with 3.65 g Fmoc-Rinkamide resin (0.59 mmol/g, Novabiochem) ina 50 ml reaction vessel fitted with a frit in the bottom (AdvancedChemTech ACT90).

Resin swelling was carried out by treating the resin with DMF (4×1min.).

The resin was deprotected using a 20% solution of piperidine in DMF (1×3min, 1×7 min, 20 ml each) and subsequently washed with DMF (1×20 ml).Acylation was carried out by addition of FmocMetOH (2.4 g, 3 eq), DMF(10 ml), HOBt (990 mg, 3 eq), and DIC (1.01 ml, 3 eq) and DMAP (260 mg,0.1 eq). The coupling was left for 4 h and the resin was washed with DMF(7×20 ml). A small sample was dried carefully and deprotected withDCM/piperidine (1:1) for 30 min. Photometric determination of theresulting Fmoc-piperidine adduct (absorption at 301 nm) gave a resinloading of 0.43 m mol/g. The remaining resin was capped using a solutionof acetic anhydride (2 M) and DMAP (0.1 M) in DMF (20 ml, 1×10 min) andsubsequently washed with DMF (12×20 ml), methanol (3×40 ml) and diethylether (3×40 ml), and dried in vacuo to yield 3.9 g FmocMet-Rinkamideresin.

10.2 Preparation of Ac-Cha-Arg-Tic-Met-NH₂ (P1) & Ac-Cha-Gpg-Tic-Met-NH₂(P67)

Peptidomimetic P1 was prepared using Fmoc solid phase synthesis startingwith FmocMet-Rinkamide resin using the same protocol as described forP51 in Example 3, but starting with a deprotection step. Coupling on Metwas performed using FmocTicOH (3 eq), TBTU (3 eq) and DIPEA (3.2 eq) for1.5 h, coupling on Tic was done using FmocArg(Pmc)OH, HOBt, DIC (3 eqeach) for 16 h, coupling on Arg was done using FmocChaOH, HOBt, DIC (3eq each) for 3 h. After the final acetylation of Cha the resin waswashed with DMF (12×7 ml), MeOH (3×20 ml), Et₂O (3×20 ml), dried invacuo and treated withTFA/water/thioanisole/1,2-ethanedithiol/triethylsilane 85.5:5:5:2.5:2for 3.5 h at r.t. The resin was filtered off, washed with TFA and theproduct was precipitated from the filtrate by adding 200 ml of chilleddiethyl ether. The suspension was kept at 0° C. for 1 h and than treatedas described in example 2.4. The crude product was purified by HPLCusing a gradient of acetonitrile in 0.1% aq TFA. MALDI-TOF MS: M/Z=659(MH+), 681 (MNa+), 697 (MK+).

Peptidomimetic P67 was prepared as P1 in Example 10 except for that theresin was coupled in coupling step 2 with FmocGpg(Pmc)OH, HOBt, DIC (1.5eq each) instead of FmocArg(Pmc)OH, HOBt, DIC (3 eq each). MALDI-TOF MS:M/Z=685.29 (MH+), 707.23 (MNa+), 723.23 (MK+).

Example 11 Preparation of Ac-Cha-Arg-Disc-Met-NH₂ (P12) &Ac-Cha-Gpg-Disc-Met-NH₂ (P66)

Peptiodomimetic P12 was prepared as described for P1 in Example 10,except for using racemic FmocDiscOH instead of FmocTicOH in couplingstep 1. The resulting two diastereomers were separated at the final HPLCstep using a gradient of acetonitrile in 0.1% aq TFA and denoted asdescribed in Example 6. MALDI-TOF MS (P12-1): M/Z=645 (MH+), 667 (MNa+).MALDI-TOF MS (P12-2): M/Z=645 (MH+), 667 (MNa+).

Peptiodomimetic P66 was prepared as described for P1 in Example 10,except for using racemic FmocDiscOH instead of FmocTicOH in couplingstep 1 and using FmocGpg(Pmc)OH, HOBt, DIC (1.5 eq each) instead ofFmocArg(Pmc)OH, HOBt, DIC (3 eq each) in coupling step 2. The resultingtwo diastereomers were separated at the final HPLC step using a gradientof acetonitrile in 0.1% aq TFA and denoted as described in Example 6.MALDI-TOF MS (P66-1): M/Z=671.25 (MH+). MALDI-TOF MS (P66-2): M/Z=671.29(MH+).

Example 12 Preparation of Ac-Cha-Arg-Tic-Met-βPhPro-NH₂ (P31) &Ac-Cha-Gpg-Tic-Met-βPhProNH₂ (P80) 12.1 Preparation ofFmoc-βPhPro-Rinkamide Resin

FmocβPhPro-Rinkamide resin was prepared by Fmoc solid phase synthesis asdescribed for FmocMet-Rinkamide resin in Example 10.1 except for usingFmocβPhProOH instead of FmocMetOH in coupling step 1.

12.2. Preparation of Ac-Cha-Arg-Tic-Met-βPhPro-NH₂ (P31) &Ac-Cha-Gpg-Tic-Met-βPhProNH₂ (P80)

Peptidomimetic P31 was prepared using Fmoc solid phase synthesisstarting with Fmoc-βPhPro-Rinkamide resin using the same protocol asdescribed for P51 in Example 3, but starting with a deprotection step.Coupling on βPhPro was done using FmocMetOH, HOBt, DIC (3 eq each) for14 h, coupling on Met was performed using FmocTicOH (3 eq), TBTU (3 eq)and DIPEA (3.2 eq) for 1.5 h, coupling on Tic was done usingFmocArg(Pmc)OH, HOBt, DIC (3 eq each) for 16 h, coupling on Arg was doneusing FmocChaOH, HOBt, DIC (3 eq each) for 3 h. After the finalacetylation of Cha the resin was treated as described in Example 10.2.The crude product was purified by HPLC using a gradient of acetonitrilein 0.1% aq TFA. MALDI-TOF MS: M/Z=832.64 (MH+).

Peptidomimetic P80 was prepared as P31 except for using FmocGpg(Pmc)OH,HOBt, DIC (1.5 eq each) instead of FmocArg(Pmc)OH, HOBt, DIC (3 eq each)in coupling step 3. MALDI-TOF MS: M/Z=859.19 (MH+), 896.14 (MK+).

Example 13 Preparation of Ac-Cha-Arg-Tic-Nle-N(Bn)CH2CH2OCH2CH2OH (P98)& Ac-Cha-Gpg-Tic-Nle-N(Bn)CH2CH2OCH2CH2OH(P101) 13.1: Preparation of2-(2-Benzyl-[2-(2-hydroxy-ethoxy)ethyl]-carbamic acid9H-fluoren-9-ylmethylester

2-(2-Aminoethoxy)-ethanol (10 ml, 100 mmol) was dissolved in dry THF (80ml). Benzaldehyde (10.5 ml, 103 mmol) was added followed by MS 4 Å andthe mixture was stirred at r.t. for 3.5 h. The resulting solution wasfiltered and concentrated under reduced pressure to yield 17.41 g oilyresidue (89 mmol). This residue was dissolved in dry THF (180 ml) andthe solution was cooled to 0° C. NaBH₄ (98 mmol) was added and themixture was stirred at r.t for 3 h. The reaction was quenched byaddition of 5 N aq. HCl (70 ml). The pH was adjusted to 11 by additionof Na₂CO₃ and the mixture was extracted with CH₂Cl₂ (4×). The combinedorganic extracts were dried over K₂CO₃, filtered and concentrated underreduced pressure to yield 15.3 g 2-(2-Benzylaminoethoxy)-ethanol, pureenough for further reaction.

Crude 2-(2-Benzylaminoethoxy)-ethanol (5.27 g, 27.0 mmol) was dissolvedin dioxane (25 ml). Na₂CO₃ (10% in water, 35 ml) was added and themixture was cooled to 0° C. After addition of FmocCl (7.81 g; 30.2 mmol)the mixture was stirred for 3.25 h. The mixture was concentrated underreduced pressure to remove dioxane. The resulting aq. mixture wasextracted with CH₂Cl₂ (3×75 ml). The combined org. extracts were driedover Na₂SO₄, filtered and concentrated under reduced pressure. Theresidue was purified by flash chromatography using ethyl acetate/hexane(1:1) as eluent to give 9.1 g of the title compound as an oil. ¹H-NMR(CDCl₃): 3.05-3.35 (m, 3H), 3.45-3.75 (m, 5H), 4.20-4.30 (m, 1H),4.45-4.55 (m, 3H), 4.69-4.68 (m, 1H), 7.00-7.45 (m, 10H), 7.55-7.80 (m,3H).

13.2: Preparation of FmocN(Bn)CH₂CH₂OCH₂CH₂O-2-chlorotrityl Resin

THF (15 ml) was added to 2-Chlorotrityl chloride resin (4.0 g, 1.2mmol/g) and the mixture was agitated for 25 min.FmocN(Bn)CH₂CH₂OCH₂CH₂OH (6.1 g, 14.7 mmol) in THF (25 ml) was addedfollowed by pyridine (850 μl, 10.5 mmol) and the mixture was heated to65° C. for 15 h. MeOH (5 ml) was added and heating was continued for 35min. The resin was filtered, washed with DMF (3×), CH₂Cl₂ (3×). MeOH(3×) and Et₂O (3×) to give 5.0 g. A small sample was dried carefully anddeprotected with DCM/piperidine (1:1) for 30 min. Photometricdetermination of the resulting Fmoc-piperidine adduct (absorption at 301nm) gave a resin loading of 0.45 mmol/g.

The peptidomimetic P98 was prepared using similar methodology to thatdescribed for Example 2.4 by deprotection and coupling usingFmocN(Bn)CH₂CH₂OCH₂CH₂O-2-chlorotrityl resin (4.93 g, 2.2 mmol),starting with a deprotection step. Coupling onHN(Bn)CH₂CH₂OCH₂CH₂O-2-chlorotrityl resin was performed using HOBt (2.5eq) DIC (2.5 eq) and FmocNleOH (2.5 eq) for 21 h. Coupling on Nle wasperformed using TBTU (3 eq), DIPEA (3.2 eq) and FmocTicOH (3 eq) in 12ml DMF with a coupling time of 1.5 h. Coupling on Tic was carried outusing FmocArg(Pmc)OH (3 eq), HOBt (3 eq) and DIC (3 eq) in 15 ml DMFover 20 h and coupling on Arg was done with FmocChaOH (3 eq), TBTU (3eq) and DIPEA (3.2 eq) in 15 ml DMF over 1.5 h. After final acetylation,resin was washed with DMF (3×), MeOH (3×) and Et₂O (3×) to give 6.67 g.The resin was treated with 50 ml ofTFA/water/thioanisole/1,2-ethanedithiol/triethylsilane 85.5:5:5:2,5:2for 3.5 h at r.t. The resin was filtered off, washed with TFA and thefiltrate was concentrated under reduced pressure. The product wasprecipitated by adding 450 ml of chilled diethyl ether to the residue.The suspension was centrifuged at 3300 rpm for 10 min, the supernatantwas discarded, the precipitate was resuspended in chilled ether,centrifuged again and the supernatant was once again discarded. Theprecipitate was dissolved in acetonitrile and 0.1% aq TFA. The organicsolvents were evaporated under reduced pressure and the aq. solution wasfreeze dried to give 1.38 g of crude product. Of this product 300 mgwere purified by HPLC using a gradient of acetonitrile in 0.1% aq TFA toyield 209 mg pure P98. MALDI-TOF MS: M(Z=819.29 (MH+).

Peptidomimetic P101 was prepared as P98 except for using FmocGpg(Pmc)OH,HOBt, DIC (1.5 eq each) instead of FmocArg(Pmc)OH, HOBt, DIC (3 eq each)in coupling step 3. MALDI-TOF MS: M/Z=845.39 (MH+), 867.38 (MNa+),883.36 (MK+).

Example 14 Competitive Binding of Compounds to MHC-II proteins DR4Dw4and DR1

Competitive binding of the peptidomimetic compounds (1 n M-100 μM) wastested on DR4Dw4 (DRA1*0101 DRB1*0401), DR1 (DRA1*0101 DRB1*0101) andDR4Dw14 (DRA1*0101 DRB1*0404) using6-(biotinamido)-hexanoyl-YAAFRAAASAKAAA-NH₂ as indicator peptidefollowing the general protocol by Ito et al. (Exp. Med. 1996; 183:2635-2644), and Siklodi et al. (Human Immunology 1998; 59: 463471) withsome modifications.

A 10 fold dilution series of compounds (4 nM-400 μM) in 25% (v/v)DMSO/50 mM sodium phosphate, 150 mM sodium chloride, pH 7.5 (PBS) wasprepared in a 96 well polypropylene plate blocked with 1% BSA/PBS for 1hours at room temperature. The MHC-compound interaction mixtures wereprepared in a similarly blocked 96 well polypropylene plate as follows;to 40 μl 2× buffer (PBS, 50 mM, pH 7.5, 2% (w/v) NP-40, 3.2 mM EDTA,6.25% protease inhibitor cocktail: 0.32 g/l of Chymostatin, Antipain,Pepstatin A, Soybean trypsin inhibitor and Leupeptin each) were given 10μl 0.8 μM indicator peptide, 20 μl compound solution of the appropriatedilution and 10 μl MHC-II DRA1*0101 DRB1*0401 (0.06 g/l), DRA1*0101DRB1*0101 (0.03 g/l) or DRA1*0101 DRB1*0404 (0.015 g/l) in 0.5% (w/v)NP-40/PBS. Interaction mixtures lacking the peptidomimetic compound andboth peptide mimetic compound and MHC-II were used as positive andnegative controls, respectively. All interaction mixtures were set up induplicates and were incubated for 16 hours at room temperature.

High binding capacity black FIA plates (Greiner, capture plates) werepreviously coated with 100 μl/well mAb LB3.1 (0.01 g/l) in PBS overnightat 4° C., and subsequently blocked with 200 μl/well 1% (w/v) BSA/PBS for1 h at room temperature. After washing with PBS, 60 μl of theMHC-II-compound interaction mixtures were transferred from theinteraction plate to the appropriate wells of the capture plate andincubated for 2 h at 4° C. The wells were washed six times with 200μl/well of cooled (4 to 8° C.) 1×DELFIA wash (Wallac-ADL-GmbH,Freiburg), incubated with 100 μl of cooled Europium-streptavidinconjugate (Wallac-ADL-GmbH, Freiburg; diluted 1/1000 with DELFIA assaybuffer) for 30 minutes at 4° C., again washed six times with cooled1×DELFIA wash and, finally, incubated with 200 μl cooled DELFIAenhancement solution (Wallac-ADL-GmbH, Freiburg) for one hour at roomtemperature before reading the time-resolved europium fluorescence atλ_(ex) Eu³⁺: 340 nm and λ_(em) Eu³⁺: 613 (615) nm (Wallac Victor² 1420Multilabel Counter, Wallac-ADL-GmbH, Freiburg).

Table 3a shows (in bold) the improved affinity of Gpg-containingcompounds of the invention to certain MHCII proteins as measured by IC50according to this example. Table 3b shows the affinities of compoundsaccording to Formula I to the same MHCII proteins. FIG. 1 shows theimproved IC₅₀ of P53 (Gpg-containing) against MHCII 0401, a preferredheptamer compound of the invention, compared to the Arg-containingpeptide (P51), and FIG. 3 shows the improved IC₅₀ of P74(Gpg-containing) against MHCII 0101, a preferred tetramer compound ofthe invention, compared to the Arg-containing peptide (P71). FIG. 3shows the improved IC₅₀ of P69 (Gpg-containing), a preferred tetramercompound of the invention, compared to the Arg-containing peptide CP82).FIG. 4 shows the improved IC₅₀ of P74 (Gpg-containing) against 0401, apreferred tetramer compound of the invention, compared to theArg-containing peptide (P71). FIG. 5 shows the improved IC₅₀ of P101(Gpg-containing), a preferred tetramer compound of the invention,compared to the Arg-containing peptide (P98).

Example 15 Competitive Binding of Compounds to MHC II Proteins Expressedon PRIESS and LG2 Cells

Competitive binding of the peptidomimetic compounds (4 nM-400 μM) wastested on Priess (DR4Dw4: DRA1*0101 DRB1*0401) and LG2 (DR1: DRA1*0101DRB1*0101) cells using 6-(biotinamido)-hexanoyl-Cha-Arg-Tic-Met-NH₂ asindicator peptide. The cells were cultured in RPMI 1640 (1×Gibco42401-042) medium, supplemented with 10% heat-inactivated FCS(Biowhittaker), 2 mM L-Glutamine, 1% non-essential amino acids stock(Gibco 11140-035; 100×MEM), 1 mM sodium pyruvate, 0.1 mg/ml Canamycinand 3.4 ppm β-mercaptoethanol. For use in the binding assay the cellswere re-suspended in medium containing 1% FCS at a density of 2.5×10⁶cells/ml.

The assay was performed in sterile 96 well polystyrene microtiterplates. A 10 fold dilution series of compounds (16 nM-1615 ™) in 1% FCSwas prepared from 5 or 10 mM compound stock solutions in 10% DMSO/water.50 μl of each compound dilution were added in duplicates to 50 μl of a16 μM solution of indicator peptide in 1% FCS. Cell binding wasinitiated by adding 100 μl of 2.5×10⁶ cells/ml 1% FCS to each well.Controls were included containing the DMSO concentration present in thesolution with the highest compound concentration. The cells wereincubated at 37° C., 6% CO₂. After 4 hours the cells were washed with200 μl PBS and lysed in 200 μl lysis buffer (50 mM sodium phosphate, 150mM sodium chloride, 1% (w/v) NP-40, 25 mM iodoacetamide, 1 mM PMSF, 3.1%protease inhibitor cocktail: 0.32 g/l of Chymostatin, Antipain,Pepstatin A, Soybean trypsin inhibitor and Leupeptin each, pH 7.5) for10 min.

High binding capacity black FIA plates (Greiner, capture plates) werepreviously coated overnight at 4° C. with 100 μl/well mAb LB3.1 (0.01g/l) in 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.5 (PBS),and subsequently blocked with 200 μl/well 1% (w/v) BSA/PBS for 1 h atroom temperature. After washing with PBS, 190 μl of cell lysate weretransferred to the appropriate wells of the capture plate and incubatedfor 2 h at 4° C. The wells were washed six times with 200 μl/well cooled(4 to 8° C.) of 1×DELFIA wash (Wallac-ADL-GmbH, Freiburg), incubatedwith 100 μl of cooled Europium-streptavidin conjugate (Wallac-ADL-GmbH,Freiburg; diluted 1/1000 with DELFIA assay buffer) for 30 minutes at 4°C., again washed six times with cooled 1×DELFIA wash and, finally,incubated with 200 μl cooled DELFIA enhancement solution(Wallac-ADL-GmbH, Freiburg) for one hour at room temperature beforereading the time-resolved europium fluorescence at λ_(ex) Eu³⁺: 340 nmand λ_(em) Eu³⁺: 613 (615) nm (Wallac Victor² 1420 Multilabel Counter,Wallac-ADL-GmbH, Freiburg).

Table 3a shows the improved affinity of Gpg-containing compounds of theinvention to certain MHCII proteins expressed on the surface of cells sameasured by IC₅₀ according to this example. Table 3b shows the affinityof compounds of Formula I towards the same protreins expressed on saidcells. FIG. 6 shows the improved IC₅₀ for inhibition of peptide bindingto MHC protein expressed on LG2 cells of P47 (Gpg-containing), apreferred heptamer compound of the invention, compared to theArg-containing peptide (P43). FIG. 7 shows the improved IC₅₀ forinhibition of peptide binding to MHC protein expressed on Priess cellsof P74 (Gpg-containing), a preferred tetramer compound of the invention,compared to the Arg-containing peptide (P71).

Example 16 Stability of Compounds in Blood Plasma

The stability of the peptidomimetic compounds was determined in rat(Charles River Laboratories, Sulzfeld), mouse (Charles RiverLaboratories, Sulzfeld) and human (Bayerisches Rotes Kreuz, München)blood plasma (E. R. Garrett and M. R. Gardner, J Pharmaceutical Sciences71 (1982) 14-25).

Table 3a shows the improved stability (in bold) of Gpg-containingcompounds of the invention in blood plasma, and FIG. 8 displays improvedstability (arbitrary units) of: a Gpg-containing heptamer compound ofthe invention (P53) in rat plasma after 24 hours compared to theArg-containing equivalent (P51); a Gpg-containing tetramer compound ofthe invention (P66-1) compared to the Arg-containing equivalent (P12-1);a Gpg-containing tetramer compound of the invention (P69 compared to theArg-containing equivalent (P82); other preferred compounds of theinvention compared to their Arg-containing equivilent. Table 3b showsimproved stability (in bold) of compounds according to Formula Icompared to compounds with an NH₂ terminating group.

Compound stock solutions were diluted into blood plasma to give a finalcompound concentration of 5 μM and the mixtures were incubated at 37° C.At 0, 6 and 24 hours 1 ml samples were drawn and the plasma proteinswere precipitated by the addition of 3 ml acetonitrile p.a. andwhirlmixing. The precipitate was pelleted by centrifugation at 2000 gfor 5 minutes. The supernatant was evaporated off to dryness and theresidue was reconstituted in 400 μl 50% acetonitrile, 0.1% TFA in water.

After filtration (0.2 μm) 200 μl of the solutions were analysed byreverse phase HPLC (PerSeptive Biosystems; Nucleosil 100-5 C18,12.5×0.46 cm; 20-50% acetonitrile in 0.1% aqueous TFA in 30 minutes),and the amount of compound remaining was estimated by integration of theappropriate peak. PBS controls were prepared for unstable compoundsaccordingly.

Example 17 Stability of Compounds Towards Degradation by Cathiepsin B1and D

The stability of the peptidomimetic compounds towards lysosomaldegradation was examined by incubation with Cathepsin B1 (EC 3.4.22.1from bovine spleen; Sigma-Aldrich, Taufkirchen; dissolved at 10000 U/lin ddH₂O) and D (EC 3.4.23.5 from bovine spleen; Sigma-Aldrich,Taufkirchen; dissolved at 10000 U/l in ddH₂O) for four hours at 37° C.using a compound concentration of 50 μM and an enzyme/substrate ratio of0.4 U/μmol. The samples contained 25 ppm 4-isopropyl benzyl alcohol(IPBA; Sigma-Aldrich, Taufkirchen) as internal standard.

Assay buffer was prepared by adding 35 μl of a 1% IPBA in DMSO solutionand 28 μl of a 10000 U/l Cathepsin B1 or Cathepsin D stock solution to14 ml of 100 mM sodium acetate, 1 mM EDTA, 1 mM DTT, pH 5.00 or 100 mMsodium acetate, pH 4.50, respectively. 8.8 μl of a 5 mM (or 4.4 μl of a10 mM) compound stock solution (in 10% DMSO/water) were placed into a1.5 ml Eppendorf tube. The reaction was started by adding 875 μl fullysupplemented assay buffer to each tube, whirlmixing and incubation at37° C. At 0 and 4 hours 400 μL samples were taken and the enzyme wasinactivated by addition of 40 μl 150% TFA aq. The samples were keptfrozen at −20° C. until analysis by RP-HPLC (200 μl injection volume;Nucleosil 100-5 C18, 12.5×4.6 cm; Gradient: 20-50% acetonitrile in 30min).

The enzyme activity of Cathepsin B1 and Cathepsin D was controlled byincubation of Ac-Cha-RAMASL-NH₂ and QYIKANSLFIGITELK, respectively,which both were completely degraded within 4 hours.

Compounds co-eluting with the internal standard during RP-HPLC analysiswere tested without the standard as Example 16.

Table 3 (a, b and c) shows the stability of compounds of the inventionagainst certain Cathepsin enzymes.

Example 18 In Vivo Inhibition of T Cell Activation from 0401(DR4) and0404(DR14) chimeric MHCII-Mice by co-Immunisation

Mouse strains DR4 and DR14 carry chimeric MHC-II transgenes that encodethe N-terminal domains of the respective DR molecules (forming thepeptide binding site) and the remaining (2nd extracellular domains,transmembrane and intracytoplasmic domains) of the murine class IImolecule I Ed. These strains are deficient of other murine class II, andthus, all helper T cell responses are triggered by peptides presented inthe respective human MHC-II binding site. As an initial in vivo test ofsubject inhibitors designed to bind to DR, DR-transgenic mice wereco-immunized with a pre-defined dose of protein antigen and differentamounts of the compound antagonist to be tested. In this set-up, bothantigen and compound were emulsified together in complete Freund'sadjuvant (CFA), and thus, a direct competition between the twocomponents for presentation by DR was tested. Readout of the assay is exvivo antigen-specific activation of T cells from regional lymph nodesexplanted 9 days after co-immunization. In a typical experiment, antigendose-response was investigated, and the curves from mice immunized withantigen were compared to those from mice co-immunized withantigen+compound. The dose response curves were generated using lymphnode cells pooled at equal numbers from 2-3 mice per experimental group.The experimental system also permits assessment of inherent antigenicityof the compound antagonist. This was done by setting up a dose-responsestudy from the same cell pool using different concentrations of thecompund under investigation instead of the antigen. As a specificitycontrol, the response to Purified Protein Derivative (PPD), the majorprotein component of CFA, was tested in the same cell pool.

FIG. 14 shows improved in-vivo inhibitory effect of a preferred tetramercompound of the invention (P69) compared to the Arg-contaiing equivilent(p82). FIG. 15 shows the in-vivo inhibitory effect of a preferredtetramer and heptamer compounds of the invention.

An estimate of overall inhibitory potential for a compund underinvestigation was taken by calculating the mean % inhibition from thecontrol over 3 concentrations of antigen; 6.7, 20 and 60 ug. Table 4shows these estimates of overall inhibitory potential of certaincompounds of the invention, together with certain Arg-containingequivalents. All Gpg conataining compounds have significantimmunosupressive properties in one or both of the in-vivo models.Gpg-containing compounds also show improved activity or improvedspecificity over the Arg-containing equivilent. Further, compoundsaccording to Formula I show activity in this assay.

18.1 Preparation of Compounds for co-Immunisation

Compounds of the invention were injected as an Emulsion prepared usingCFA (Bacto Adjuvant Complete H37 Ra, Difco, Order-No. 3113-60); proteins10 mg/ml in PBS, inhibitors 5 μM in 10% DMSO. CFA, PBS, antigen, andinhibitor were placed in the barrel of a 5-ml syringe (total volume notmore than 1 mL). The mixture was sonicated at an amplitude less than 40%(30-35%) for two separated 15 s periods. The emulsion should seem to behard and not liquid at this point. A drop of emulsion placed on thesurface of water should not melt. The resulting emulsion was pushedthrough the needle into a 1 ml syringe with a needle (Gr.18, 26G×1″),and air bubbles were removed. Mice were co-immunized with 50 μg proteinand 210 nM compound antagonist in final 100 μl emulsion into the tailbase. Antigen+Compound and CFA are mixed 1:1 vol/vol. The best resultsare obtained using DR 4 mice immunised with HEL and DR 14 mice immunizedwith OVA.

18.2 Immunisation of Mice

Mice were injected at the tail-base as follows: Holding the tail of amouse with thumb and middle finger, the index finger was placed underthe base of the mouse's tail. The needle of the syringe was insertedabout 1.5 cm from the tail base (hairy end) under the skin and pushedabout 1 cm into the tail. 100 μL emulsion was injected into the mouse.After 9 days, mice were killed and lymph nodes removed.

18.3 T Cell Proliferation Assay After Co-Immunisation

The inhibitory effect of the candidate compounds on T-cell activationwas tested using T-cells and antigen-presenting cells isolated from thelymph nodes of chimeric DR4-IE transgenic mice (Taconic, USA) previouslyco-immunized with hen egg lysozyme plus compound, or DR14-IE transgenicmice co-immunized with ovalbumin plus compound according to standardprocedures (Adorini et al., 1988, Mueller et al., 1990; CurrentProtocols in Immunology, Vol. 2, 7.21; Ito et al., 1996).

About 9 days (e.g., 8-10 days) after immunisation, mice were killed andlymph nodes removed, a T cell proliferation assay was performedaccording to Example 19.1, using increasing amounts of antigen on lymphnode cells from mice that had been co-immunised with protein antigen andcompound antagonist. The control line was immunised without compound.

Antigen dilutions were prepared and distributed on a 96-U-Well-MTPaccording to the assay plan below. PPD serves as a positive control forT-cell proliferation.

Medium (Negative Control)

-   -   100 μl HL-1    -   100 μl cells (2.5×10⁵ cells)        PPD (Positive Control)    -   100 μl PPD solution (100 μg/ml))    -   100 μl cells (2.5×105 cells)        Inhibitor Control    -   100 μl Compound solution (100-3.125 μM)    -   100 μl cells (2.5×105 cells)        Antigen-Titration (600 μg/ml-0.82 μg/ml)    -   100 μl antigen solution    -   100 μl cells (2.5×105 cells)        Wash Medium    -   97.5% RPMI    -   1.5% FCS    -   1% Kanamycin (Stock solution 10 mg/ml 4 final 0.1 mg/ml)        HL-1 Medium    -   98% HL-1 Medium (BioWittaker Europe Order-no. 77201)    -   1% L-Glutamin (Stocksolution 200 mM→final 2 mM) 1% Kanamycin        (Stocksolution 10 mg/ml→final 0.1 mg/ml) no serum        Stock Solutions    -   HEL (Lysozyme Grade m: From Chicken Egg White; Sigma L-7011) 10        mg/ml PBS    -   OVA (Albumine, Chicken Egg; Sigma A-2512) 4 mg/ml PBS    -   PPD (Tuberculin PPD; Statens Serum Institut; Order-no. 2391) 1        mg/ml (ready for use)        Solutions for Use    -   HEL: Dilute stock solution 1:83 with HL-1 medium (120 μg/ml) 4        final concentration in well 30 μg/ml.    -   OVA: Dilute stock solution 1:48 with HL-1 medium (84        μg/ml)→final 21 μg/ml.    -   PPD: Dilute stock solution 1:10 with HL-1 medium (100        μg/ml)→final 25 μg/ml.

Example 19 In Vitro Inhibition of T Cell Activation by Compounds of theInvention

Immunomodulatory properties of compounds under investigation were testedusing an assay that measures T cell proliferation. Table 3 (a and b)shows the IC50 (uM) and maximal inhibition (%) of certain compounds.FIG. 9 displays a dose-response curve demonstrating improvedimmunosuppressive properties as measured by a T-cell activation assay ofP53 (Gpg-containing), a preferred heptamer compound of the invention,compared to the Arg-containing peptide (P51). A dose-response curve ofanother compound of the invention (P41-1) is also shown. FIG. 10displays a dose-response curves demonstrating the immunosuppressiveproperties as measured by a T-cell activation assay of various preferredcompounds of the invention; P69, P74, P101 and P53.

The compounds were tested as follows to inhibit the proliferative T cellresponse of antigen-primed lymph node cells from mice carrying achimeric mouse-human class II transgene with an RA-associated peptidebinding site, and lack murine class II molecules (Muller et al., 1990;Woods et al., 1994; Current Protocols in Immunology, Vol. 2, 7.21; Itoet al., 1996). Here, the immunization takes place in vivo, but theinhibition and readout are ex vivo. Transgenic mice expressing MHC classII molecules with binding sites of the RA associated molecule, DRB*0401were commercially obtained. These mice lack murine MHC class II, andthus, all Th responses are channelled through a single humanRA-associated MHC class II molecule (Ito et al. 1996). These transgenicmice represent a model for testing human class II antagonists.

19.1 T Cell Proliferation Assay

The inhibitory effect of the compounds under investigation were testedon T-cell proliferation measured using chimeric T-cells and antigenpresenting cells isolated from the lymph nodes of chimeric 0401-IEtransgenic mice (Taconic, USA) previously immunized with hen eggovalbumin (Ito et al. 1996) according to standard procedures. 1.5×10⁵cells are incubated in 0.2 ml wells of 96-well tissue culture plates inthe presence of ovalbumin (30 μg per well—half-maximal stimulatoryconcentration) and a dilution series of the compound under test (fromaround 0.1 to 200 uM) in serum free HL-1 medium containing 2 mML-glutamine and 0.1 g/l Kanamycin for three days. Antigen specificproliferation is measured by 3H-methyl-thymidine (1 μCi/well)incorporation during the last 16 h of culture (Falcioni et al., 1999).Cells are harvested, and 3H incorporation measured using a scintillationcounter (TopCount, Wallac Finland). Inhibition of T-cell proliferationon treatment with the compound may be observed by comparison to controlwells containing antigen.

Example 20 Inhibition of IL-2 Secretion Front T-Cell Hybridoma Cells byCompounds of the Invention

The Gpg-containing compounds of the invention displayed substantialimmunomodulatory properties within an assay measuring IL-2 secretionfrom immortalized T-cells. Table 3a shows the IC₅₀ (uM) and maximalinhibition (%) of Gpg compounds in this assay. Table 3b shows improvedactivity (in bold) of tetramer compounds according to Formula I comparedto those containing an NH₂ terminating group. FIG. 11 displays adose-response curve demonstrating the improved immunosuppressiveproperties as measured by IL-2 secretion of P41-1 (Gpg-containing), aheptamer compound of the invention, compared to the Arg-containingpeptide (P40-1), and FIG. 12 displays a dose-response curvedemonstrating the improved immunosuppressive properties as measured byIL-2 secretion of P69 (Gpg-containing), a tetramer compound of theinvention, compared to the Arg-containing peptide (P82). FIG. 13 showsthe immunosuppressive properties of P53 (Gpg-containing), a preferredheptamer compound of the invention, compared to a DMSO control.

The immunomodulatory properties of the compounds under investigation wasinvestigated by measuring IL-2 secretion from the hybridoma cell lineT-Hyb 1 stimulated using DR-transgenic antigen presenting cells (APC)under conditions of half-maximal antigen stimulation. IL-2 secretion wasdetected and measured using a standard ELISA method provided by theOptiEIA mouse IL-2 kit of Pharmingen (Torrey Pine, Calif., USA). APCswere isolated from the spleen of unimmunized chimeric 0401-IE transgenicmice ato et al. 1996) according to standard procedures. 1.5×10⁵ APCswere added to 0.2 ml wells of 96-well in RPMI medium containing thefollowing additives (all from Gibco BRL and PAA): 10% FCS, 2 mML-glutamine, 1% non-essential amino acids, 1 mM sodium pyruvate and 0.1g/l kanamycin. Hen egg ovalbumin was added to a final concentration of200 μg/ml in a final volume of 100 μl of the above medium, the cellsincubated with this antigen for 30 m in at 37° C. under 6% C O₂.Compounds were added to each well at various concentrations (typicallyin a range from 0.1 to 200 μM), the plate incubated for 1 h at 37° C./6%CO₂ and 2×10⁵ T-Hyb 1 cells added to give a final volume of 200 μl inthe above medium. After incubation for 24 h, 100 μl of supernatant wastransferred to an ELISA plate (Nunc-Immuno Plate MaxiSorp surface, Nunc,Roskilde, DK) previously coated with IL-2 Capture Antibody (BDPharmingen, Torrey Pine, Calif., USA), the amount of IL-2 was quantifiedaccording to the manufacturer's directions using the OptiEIA Mouse IL-2kit and the plate read using a Victor V reader (Wallac, Finland).Secreted IL-2 in pg/ml was calibrated using the IL-2 standards providedin the kit.

The T-cell hybridoma line T-Hyb1 was established by fusion of a T-cellreceptor negative variant of the thymoma line BW 5147 (ATCC) and lymphnode cells from chimeric 0401-IE transgenic mice previously immunizedwith hen egg ovalbumin (Ito et al. 1996). The clone T-Hyb1 was selectedfor the assay since it responded to antigen specific stimulation withhigh IL-2 secretion.

Example 21 Immunomodulatory Activity of Compounds of the InventionWithin Mouse Disease Models

Compounds showing the best profile (binding affinity, proteasestability, T cell inhibition in vitro and in vivo) are tested for theirtherapeutic potential in MHC-II transgenic mouse models of autoimmunediseases, namely collagen induced arthritis (CIA)—a model for rheumatoidarthritis; and experimental autoimmune encephalomyelitis (EAE)—a modelfor multiple sclerosis.

CIA is induced by immunization with type II collagen in HLA-DR1transgenic mice as described by Rosloniec et al (J. Exp. Med. 1997; 185:113). After disease onset, mice are treated with compounds at themaximal tolerated dose s.c. for two weeks, and the disease developmentcompared to that in mice treated with solvent only as follows: DayTreatment 1 Immunization (bovine C-II + CFA) 19-40 Compound injecteds.c. starting at disease onset 5× per week for 3 weeks

Disease Severity Score

-   -   1. Erythema and mild swelling confined to the tarsals or ankle        joint    -   2. Erythema and mild swelling extending from the ankle to the        tarsals    -   3. Erythema and moderate swelling extending from the ankle to        the metatarsal joints    -   4. Erythema and severe swelling encompass the ankle, foot, and        digits

FIG. 16 shows the efficacy of preferred compounds of the invention (P96,P53 and P74) in the CIA mouse model for rheumatoid arthritis compared tosolvent as control.

EAE is induced in DR4 transgenic mice by injection of myelinoligodendrocyte glycoprotein (MOG) as described by Ito et al. (1996).After disease onset, mice are treated with compounds and studied asbelow: Day Treatment 0 Immunization (MOG + CFA) 14-36 Compound injecteds.c. starting at disease induction or onset 5× per week

Disease Score

-   -   1. Tail atony    -   2. Hind limb weakness    -   3. Hind limb paralysis    -   4. Hind limb paralysis and fore limb weakness or paralysis    -   5. Moribund

FIG. 17 shows the efficacy of preferred compounds of the invention (P69,P53 and P74) in the EAE mouse model for multiple sclerosis preventioncompared to solvent as control.

FIG. 18 shows the efficacy of preferred compounds of the invention (P69and P53) in the EAE mouse model for multiple sclerosis treatmentcompared to solvent as control.

FIG. 19 shows the efficacy of a preferred Gpc-containing compound of theinvention (P69) compared to equivilent Arg containing compound (P82) inthe EAE mouse model of multiple sclerosis. The efficacy of the Gpgcompound is superior in terms of disease treatment than the Argcompound, despite being treated at half concentration (125 nM) of theArg compound (250 nM).

Example 22 Pharmaceutical Compositions

In order to select the most appropriate compound of the invention toenter further experiments and to assess its suitability for use in atherapeutic composition for the treatment of diseases of the immunesystem, additional data are collected. Such data for each compund caninclude the affinity, reactivity, specificity, IC50-values, forinhibition of IL-2 secretion and of T-cell proliferation, as estimatedin vitro, and DR-transgenic models of rheumatoid arthritis, and multiplesclerosis.

The activity of compounds of the invention may be compared againstpreviously accepted therapies or theraputics for a given disorder. Forexample, a particular compound of the invention may be compared againstInterferon-beta, an accepted therapy for multiple sclerosis.

The compound that shows appropriate affinity, best specificity and/orgreatest inhibition of T-cell proliferation or IL-2 secretion, and highefficacy in inhibiting rheumatoid arthritis, and multiple sclerosis inappropriate models, might be chosen to enter further experiments. Suchexperiments may include, for example, therapeutic profiling andtoxicology in animals and phase I clinical trials in humans.

The compounds of the invention may be administered for therapeutic orprophylactic use to warm-blooded animals such as humans in the form ofconventional pharmaceutical compositions, a typical example of whichincludes the following: Injectable Solution: 0.01 to 100 mg of activeingredient is dissolved in up to 2 mL of an aqueous injection vehicle togive a concentration of active ingredient between 0.01 to 100 mg/mL. Theaqueous injection vehicle is buffered to a pH between 5 and 8, asneeded, using a pharmaceutically acceptable buffer (for example,phosphate or acetate) and contains a pharmaceutically acceptabletonicity adjustment agent (for example, NaCl or dextrose) added toachieve isotonicity. The vehicle may optionally also contain otherpharmaceutically acceptable excipients such as solubilizing agents (forexample, DMSO, ethanol, propylene glycol, polyethylene glycol, etc.)preservatives, and antioxidants. The active ingredient may typically bea compound described hereinabove and may conveniently be present as apharmaceutically acceptable salt. The compound of the invention may beadministered together with one or more other active ingredients. Suchcompositions may be a single package, pill, or application containingseveral such active ingredients, or such administration may comprisesequential or repeated administrations of the separate activeingredients that include a compound of the invention.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thecompounds and methods of use thereof described herein. Such equivalentsare considered to be within the scope of this invention and are coveredby the following claims.

All patents, published patent applications, and publications citedherein are incorporated by reference as though set forth fully herein.TABLE 1 Gpg-containing compounds of the invention # Pos. 2 Sequence a.Preferred Gpg-containing compounds of the invention. P41-1 GpgAc-Cha-Gpg-Disc-Met-βPhPro-[S(oxaz)L]-NMe2 P41-2 GpgAc-Cha-Gpg-Disc-Met-βPhPro-[S(oxaz)L]-NMe2 P45-1 GpgAc-Cha-Gpg-Disc-Met-βPhPro-OH P45-2 Gpg Ac-Cha-Gpg-Disc-Met-βPhPro-OHP47 Gpg Ac-Cha-Gpg-Tic-Met(O)-βPhPro-[S(oxaz)L]-NMe2 P52 GpgAc-Phe-Gpg-Tic-Met(O)-βPhPro-(S(oxaz)L]-NMe2 P53 GpgAc-Cha-Gpg-Tic-Nle-βPhPro-(S(oxaz)L]-NMe2 P54 GpgAc-Cha-Gpg-Tic-Nle-βPhPro-N(Me)CH2CH2OH P55 GpgAc-Phe-Gpg-Tic-Nle-βPhPro-[S(oxaz)L]-NMe2 P56 GpgAc-Phe-Gpg-Tic-Nle-βPhPro-N(Me)CH2CH2OH P57 GpgAc-Hfe-Gpg-Tic-Nle-βPhPro-[S(oxaz)L]-NMe2 P58 GpgAc-Thi-Gpg-Tic-Nle-βPhPro-[S(oxaz)L]-NMe2 P59 GpgAc-Cha-Gpg-Tic-Ile-βPhPro-[S(oxaz)L]-NMe2 P60 GpgAc-Cha-Gpg-Tic-Met-βPhPro-[S(oxaz)L]-NMe2 P61-1 GpgAc-Cha-Gpg-Disc-Nle-βPhPro-[S(oxaz)L]-NMe2 P61-2 GpgAc-Cha-Gpg-Disc-Nle-βPhPro-[S(oxaz)LI-NMe2 P62-1 GpgAc-Phe-Gpg-Disc-Met-βPhPro-[S(oxaz)L]-NMe2 P62-2 GpgAc-Phe-Gpg-Disc-Met-βPhPro-[S(oxaz)L]-NMe2 P63-1 GpgAc-Thi-Gpg-Disc-Met-βPhPro-[S(oxaz)L]-NMe2 P63-2 GpgAc-Thi-Gpg-Disc-Met-βPhPro-[S(oxaz)L]-NMe2 P64 GpgAc-Cha-Gpg-Disc-Met(O)-βPhPro-[S(oxaz)L]-NMe2 P65 GpgAc-Thi-Gpg-Disc-Met(O)-βPhPro-[S(oxaz)L]-NMe2 P66-1 GpgAc-Cha-Gpg-Disc-Met-NH2 P66-2 Gpg Ac-Cha-Gpg-Disc-Met-NH2 P67 GpgAc-Cha-Gpg-Tic-Met-NH2 P68 Gpg Ac-Cha-Gpg-Tic-Nle-N(H)Bn P69 GpgAc-Cha-Gpg-Tic-Nle-N(H)CH2CH2Ph P70 GpgAc-Cha-Gpg-Tic-Nle-N(H)CH2CH2OCH2CH2OH P74 GpgAc-Cha-Gpg-Tic-Nle-N(Me)Bn P76-1 Gpg Ac-Cha-Gpg-Disc-Nle-N(Me)Bn P76-2Gpg Ac-Cha-Gpg-Disc-Nle-N(Me)Bn P77 GpgAc-Cha-Gpg-Tic-Nle-tetrahydroisoquinoline P78 GpgAc-Cha-Gpg-Tic-Nle-N(Bn)CH2CH2OH P80 Gpg Ac-Cha-Gpg-Tic-Met-(βPhProNH2P101 Gpg Ac-Cha-Gpg-Tic-Nle-N(Bn)CH2CH2OCH2CH2OH P102 GpgAc-Cha-Gpg-Tic-Met-N(Bn)CH2CH2OCH2CH2OH Prefered substitutions Leadpeptide Ac (Cha) R A M A S L -NH₂ b. Preferred mimetic substitutions ofthe lead natural peptide (Falcioni et al; 1999) according to Formula II.Ac Cha Gpg Ala Met Ala Ser Leu -NH₂ 4-aminobutyryl Nba C(Acm) Nleβ-PhPro tLeu 3-aminopropyl 4-MeCha C(Prm) Chg--------N(H)CH(CH₂OH)₂₋₋₋₋₋₋₋₋ Coa MePhg ---Haic-----------N(CH₃)CH₂CH₂OH-------- Hfe C(Ac) --Odapdc-----------N(H)CH₂CH₂OH--------- Phe Nva Met(O)----[S-Ψ(oxaz)-L]-N(CH₃)₂₋₋₋₋₋ Tic Ile ----[S-Ψ(imid)-L]-N(CH₃)₂₋₋₋₋₋Disc ----------N(H)CH₂tBu---------- Thiq -----------D-Leu-ol-----------azaTic -----------D-Leu-ol----------- ------------N(H)Bn-----------------------N(CH3)Bn----------- ---------N(H)CH2CH2Ph-----------------N(CH3)CH2CH2Ph-------- --------N(CH3)CH2CH2OH-----------------N(Bn)CH2CH2OH-------- ------N(CH2CH2Ph)CH2CH2OH----------N(H)CH2CH2OCH2CH2OH------ -----N(Bn)CH2CH2OCH2CH2OH-------N(CH2CH2Ph)CH2CH2OCH2CH2OH-- ----tetrahydroisoquinoline--------------isoindoline--------- Prefered substitutions Lead peptide Ac(Cha) R A M A S L -NH₂ c. Preferred tetramer mimetic substitutions ofthe lead natural peptide (Falcioni et al; 1999) according to Formula I.Ac Cha Gpg Ala Met -----------------N(H)Bn-----------------4-aminobutyryl Nba Arg C(Acm) Nle----------------N(CH₃)Bn---------------- 3-aminopropyl 4-MeCha OrnC(Prm) Chg --------------N(H)CH₂CH₂Ph-------------- Coa 4-Pya MePhg Ile-------------N(CH₃)CH₂CH₂Ph------------- Hfe αMeOrn C(Ac) Met(O)-------------N(CH₃)CH₂CH₂OH------------- Phe Cit Nva-------------N(Bn)CH₂CH₂OH-------------- alle Tic----------N(CH₂CH₂Ph)CH₂CH₂OH----------- Val Disc----------N(H)CH₂CH₂OCH₂CH₂OH----------- 3-Pya Thiq---------N(Bn)CH₂CH₂OCH₂CH₂OH----------- ---Odapdc-----------N(CH₂CH₂Ph)CH₂CH₂OCH₂CH₂OH-------- azaTic---------tetrahydroisoquinoline----------------------isoindoline----------------

TABLE 2 Other compounds investigated with the assays described herein #Pos. 2 Sequence Arg Ac-Cha-Arg-Tic-Haic-[S(oxaz)L]-N(CH3)2 ArgAc-Cha-Arg-Tic-Haic-NH2 Arg Ac-Cha-Arg-Tic-Haic-Ser-MeLeu-NH2 ArgAc-Cha-Arg-Tic-Met-βPhPro-Ser-tLeu-NH2 P1 Arg Ac-Cha-Arg-Tic-Met-NH2 P3Arg Ac-Cha-RAMASL-NH2 P6 Arg Ac-Cha-Arg-Thiq-Met-NH2 P8 OmAc-Cha-Om-Tic-Met-NH2 P9 Om Ac-Cha-Om-Thiq-Met-NH2 P10 ValAc-Cha-Val-Tic-Met-NH2 P11 Val Ac-Cha-Val-Thiq-Met-NH2 P12-1 ArgAc-Cha-Arg-Disc-Met-NH2 P12-2 Arg Ac-Cha-Arg-Disc-Met-NH2 P13 alleAc-Cha-alle-Tic-Met-NH2 P14 alle Ac-Cha-alle-Thiq-Met-NH2 P15-1 ValAc-Cha-Val-Disc-Met-NH2 P15-2 Val Ac-Cha-Val-Disc-Met-NH2 P16-1 OmAc-Cha-Om-Disc-Met-NH2 P16-2 Om Ac-Cha-Om-Disc-Met-NH2 P17-1 alleAc-Cha-alle-Disc-Met-NH2 P17-2 alle Ac-Cha-alle-Disc-Met-NH2 P18-1 OmAc-Cha-Om-azaTic-Met(O)-NH2 P18-2 Om Ac-Cha-Om-azaTic-Met-NH2 P20 ArgAc-Cha-Arg-azaTic-Met-NH2 P21 Arg Ac-Cha-Arg-D-Tic-Met-NH2 P22 CitAc-Cha-Cit-Disc-Met-NH2 P24 Cit Ac-Cha-Cit-D-Tic-Met-NH2 P25 CitAc-Cha-Cit-Tic-Met-NH2 P26 Om Ac-Cha-Om-D-Tic-Met-NH2 P28 “Arg”Ac-YGRKKRRQRRR-(ACS)Cha-R-Tic-M-NH2 P30 Arg Ac-Cha-Arg-Tic-Met-βPhPro-OHP31 Arg Ac-Cha-Arg-Tic-Met-βPhPro-NH2 P32 3Pya Ac-Cha-3Pya-Tic-Met-NH2P33 Arg Ac-Cha-Arg-Tic-Met-βPhPro-[S(oxaz)L]-NMe2 P34 4PyaAc-Cha-4Pya-Tic-Met-NH2 P35-1 aMeOm Ac-Cha-αMeOm-Tic-Met-NH2 P35-2 aMeOmAc-Cha-αMeOm-Tic-Met-NH2 P36 Arg Ac-Cha-Arg-(N,N¹-Et2)-Tic-Met-NH2 P38-2Arg Ac-Cha-Arg-(Et2)-Disc-Met-NH2 P39 OmAc-Cha-Om-Tic-Met-βPhPro-[S(oxaz)L]NMe2 P40-1 ArgAc-Cha-Arg-Disc-Met-βPhPro-[S(oxaz)L]NMe2 P40-2 ArgAc-Cha-Arg-Disc-Met-βPhPro-[S(oxaz)L]-NMe2 P42 Arg H-Cha-Arg-Tic-Met-NH2P43 Arg Ac-Cha-Arg-Tic-Met(O)-βPhPro-[S(oxaz)L]-NMe2 P44-1 ArgAc-Cha-Arg-Disc-Met-βPhPro-N(Me)CH2CH2OH P44-2 ArgAc-Cha-Arg-Disc-Met-βPhPro-N(Me)CH2CH2OH P48 N/AO,O′-Bis-(CH2-CH2-NH-CO-Cha-Arg-Tic-Met-NH2)-PEG 3400 P49 ArgAc-Phe-Arg-Tic-Met-NH2 P50 Arg Ac-Cha-Arg-Tic-Ile-NH2 P51 ArgAc-Cha-Arg-Tic-Nle-βPhPro-[S(oxaz)L]-NMe2 P71 ArgAc-Cha-Arg-Tic-Nle-N(Me)Bn P72-1 Arg Ac-Cha-Arg-Disc-Nle-N(Me)Bn P72-2Arg Ac-Cha-Arg-Disc-Nle-N(Me)Bn P73 Arg H-Cha-Arg-Tic-Nle-NMe2 P75-1 ArgAc-Cha-Arg-Hbc-Nle-N(Me)Bn P75-2 Arg Ac-Cha-Arg-Hbc-Nle-N(Me)Bn P79 ArgAc-Cha-Arg-Phe-Nle-N(Me)Bn P81 ArgAc-Cha-Arg-Tic-Nle-N(CH2CH2OH)CH2CH2Ph P82 ArgAc-Cha-Arg-Tic-Nle-N(H)CH2CH2Ph P83 Arg Ac-Cha-Arg-Tic-Nle-N(Me)CH2CH2PhP84 Arg Ac-Cha-Arg-Tic-Met-N(Me)CH2CH2Ph P85 ArgAc-Cha-Arg-Tic-Met-N(CH2CH2OH)CH2CH2Ph P86 ArgAc-Cha-Arg-Tic-Nle-N(CH2CH2Ph)CH2CH2OCH2CH2OH P89 ArgAc-Cha-Arg-Tic-NBu2 P90 Arg Ac-Cha-Arg-Tic-Gly-N(Me)Bn P91 ArgAc-Cha-Arg-Tic-Aib-N(Me)Bn P92 Arg Ac-Cha-Arg-Tic-Nle-N(Me)CH2CH2OH P93Arg Z-Cha-Arg-N(Me)Bn P94 Arg Ac-Cha-Arg-Tic-Aib-N(Bn)CH2CH2OH P95 ArgAc-Cha-Arg-Tic-Gly-N(Bn)CH2CH2OH P96 ArgAc-Cha-Arg-Tic-Met-N(CH2CH2Ph)CH2CH2OCH2CH2OH P97 ArgAc-Cha-Arg-Tic-N(H)Bu P98 Arg Ac-Cha-Arg-Tic-Nle-N(Bn)CH2CH2OCH2CH2OHP99 Arg Ac-Cha-Arg-Tic-Met-N(Bn)CH2CH2OCH2CH2OH P100 ArgAc-Cha-Arg-Tic-NH(C5H11)

TABLE 3 a. Biological properties of certain compounds of the inventionwith reference to Formula II Protein binding Cell binding IC50 (nM) IC50(uM) Number Pos. 2 Sequence 0401 0101 0404 Priess (0401) LG2 (0101) P33Arg Ac-Cha-Arg-Tic-Met-βPhPro-[Sψ(oxaz)L]-NMe₂ 300 190 220 3.2 4.6 P60Gpg Ac-Cha-Gpg-Tic-Met-βPhPro-[Sψ(oxaz)L]-NMe₂ 230 250 190 2.1 1.5 P51Arg Ac-Cha-Arg-Tic-Nle-βPhPro-[Sψ(oxaz)L]-NMe₂ 670 400 210 NT 3 P53 GpgAc-Cha-Gpg-Tic-Nle-βPhPro-[Sψ(oxaz)L]-NMe₂ 410 340 180 3 3.5 P43 ArgAc-Cha-Arg-Tic-Met(O)-βPhPro-[Sψ(oxaz)L]-NMe₂ 320 380 1,700 3.1 13 P47Gpg Ac-Cha-Gpg-Tic-Met(O)-βPhPro-[Sψ(oxaz)L]-NMe₂ 180 330 580 6.9 4.6P40-1 Arg Ac-Cha-Arg-Disc-Met-βPhPro-[Sψ(oxaz)L]-NMe₂ 270 140 98 3 8P41-1 Gpg Ac-Cha-Gpg-Disc-Met-βPhPro-[Sψ(oxaz)L]-NMe₂ 200 160 110 3.73.8 P82 Arg Ac-Cha-Arg-Tic-Nle-N(H)CH₂CH₂Ph 18,000 3,100 14,000 NT NTP69 Gpg Ac-Cha-Gpg-Tic-Nle-N(H)CH₂CH₂Ph 3,600 690 4,500 11 8.2 P12-1 ArgAc-Cha-Arg-Disc-Met-NH₂ 370 430 860 6.7 9.6 P66-1 GpgAc-Cha-Gpg-Disc-Met-NH₂ 290 290 590 7.8 6 P72-1 ArgAc-Cha-Arg-Disc-Nle-N(Me)Bn 5,200 1,200 1,700 7.5 12 P76-1 GpgAc-Cha-Gpg-Disc-Nle-N(Me)Bn 2,300 550 1,400 9.8 4.6 P1 ArgAc-Cha-Arg-Tic-Met-NH₂ 1,600 260 2,300 11 6.8 P67 GpgAc-Cha-Gpg-Tic-Met-NH₂ 410 120 1,060 4.5 4.5 P71 ArgAc-Cha-Arg-Tic-Nle-N(Me)Bn 11,000 860 6,600 11 6.9 P74 GpgAc-Cha-Gpg-Tic-Nle-N(Me)Bn 3,400 360 2,200 9.2 6.1 P31 ArgAc-Cha-Arg-Tic-Met-βPhPro-NH₂ 280 290 850 3.7 8.5 P80 GpgAc-Cha-Gpg-Tic-Met-βPhProNH₂ 670 550 470 2.7 2.7 P98 ArgAc-Cha-Arg-Tic-Nle-N(Bn)CH₂CH₂OCH₂CH₂OH 4,700 640 1,300 NT NT P101 GpgAc-Cha-Gpg-Tic-Nle-N(Bn)CH₂CH₂OCH₂CH₂OH 1,500 500 650 NT NT P99 ArgAc-Cha-Arg-Tic-Met-N(Bn)CH2CH2OCH2CH2OH 3,000 560 1,900 NT NT P102 GpgAc-Cha-Gpg-Tic-Met-N(Bn)CH2CH2OCH2CH2OH 1,700 580 2,100 NT NTImmunsupressive assays Stability (Arbitrary units) IC50 (uM) and Max %Inhibition) Rat, Mouse or Human plasma; Cathepsins DR4-LN DR4-LN DR14-LNDR14-LN Thyb1 Thyb1 Rat Rat Number (IC50) (Max) (IC50) (Max) (IC50)(Max) 6h 24 h P33 NT NT NT NT 28 91 78 40 P60 29 97 41 98 30 47 134 124P51 36 100 52 99 62 88 81 60 P53 40 100 32 101 25 99 114 108 P43 NT NTNT NT 54 84 96 71 P47 77 77 NT NT 62 83 116 103 P40-1 NT NT NT NT 59 81111 71 P41-1 49 96 25 98 47 83 91 103 P82 27 100 NT NT 78 100 65 18 P6919 101 23 107 42 96 41 42 P12-1 >200 5 NT NT NT NT 67 10 P66-1 120 20 NTNT 100 24 138 81 P72-1 31 103 29 95 88 105 80 67 P76-1 34 103 NT NT 64106 80 84 P1 84 41 NT NT >200 NM 11 0 P67 NT NT NT NT 115 33 10 34 P7119 102 15 96 78 101 89 53 P74 18 102 14 97 47 103 91 71 P31 18 64 98 8955 64 42 5 P80 NT NT 94 88 13 100 102 39 P98 69 67 >200 NM 79 86 71 60P101 66 100 46 100 57 88 73 81 P99 120 50 105 100 97 62 80 62 P102 11060 73 100 81 76 131 121 Stability (Arbitrary units) Rat, Mouse or Humanplasma; Cathepsins Human Human Mouse Mouse Cath B Cath D Cath L Number 6h 24 h 6 h 24 h 4 h 4 h 4 h P33 97 94 97 69 98 97 107 P60 88 120 111 9598 108 NT P51 119 129 118 136 103 103 NT P53 110 122 117 115 97 99 NTP43 134 131 96 82 96 101 NT P47 126 140 126 111 100 99 NT P40-1 108 103NT NT 99 93 NT P41-1 59 115 100 111 104 103 NT P82 94 72 79 31 105 106NT P69 105 93 115 85 92 100 NT P12-1 75 0 76 38 96 103 101 P66-1 103 87115 112 102 101 NT P72-1 99 99 97 75 98 101 NT P76-1 93 107 93 16 100104 NT P1 94 85 NT 5 87 102 117 P67 97 100 71 16 58 113 NT P71 107 90117 87 97 104 NT P74 114 125 146 97 105 104 NT P31 93 86 86 56 102 95103 P80 139 134 106 123 90 84 NT P98 116 121 87 54 104 104 NT P101 106107 107 94 100 106 NT P99 104 98 71 13 106 107 NT P102 110 116 105 101116 120 NT b. Biological properties of certain compounds of theinvention with reference to Formula I Protein binding IC50 (nM) NumberPos. 2 Sequence 0401 0101 0404 P67 4 NH2 Ac-Cha-Gpg-Tic-Met-NH₂ 410 1201,080 P102 4 N(Bn)CH2CH2OCH2CH2OH Ac-Cha-Gpg-Tic-Met- 1,700 580 2,100N(Bn)CH2CH2OCH2CH2OH P60 7 NMe2 Ac-Cha-Gpg-Tic-MetβPhPro- 230 250 190[SΨ(oxaz)L]-NMe₂ P1 4 NH2 Ac-Cha-Arg-Tic-Met-NH₂ 1,600 260 2,300 P99 4N(Bn)CH2CH2OCH2CH2OH Ac-Cha-Arg-Tic-Met- 3,000 690 1,900N(Bn)CH2CH2OCH2CH2OH P85 4 N(CH2CH2OH)CH2CH2Ph Ac-Cha-Arg-Tic-Met-13,000 1,800 31,000 N(CH2CH2OH)CH2CH2Ph P96 4 N(CK2CH2Ph)CH2CH2OCH2CH2OHAc-Cha-Arg-Tic-Met- 5,900 1,000 7,400 N(CH2CH2Ph)CH2CH2OCH2CH2OH P84 4N(Me)CH2CH2Ph Ac-Cha-Arg-Tic-Met- 7,900 900 14,000 N(Me)CH2CH2Ph P33 7NMe2 Ac-Cha-Arg-Tic-Met- 300 190 220 βPhPro-[SΨ(oxaz)L]-NMe₂ P69 4N(H)CH2CH2Ph Ac-Cha-Gpg-Tic-Nle- 3,600 690 4,500 N(H)CH₂CH₂Ph P101 4N(Bn)CH2CH2OCH2CH2OH Ac-Cha-Gpg-Tic-Nle- 1,500 500 650N(Bn)CH₂CH₂OCH₂CH₂OH P74 4 N(Me)Bn Ac-Cha-Gpg-Tic-Nle-N(Me)Bn 3400 3602,200 P78 4 N(Bn)CH2CH2OH Ac-Cha-Gpg-Tic-Nle- 2,600 430 1,200N(Bn)CH2CH2OH P68 4 N(H)Bn Ac-Cha-Gpg-Tic-Nle-N(H)Bn 2,100 600 2,000 P704 N(H)CH2CH2OCH2CH2OH Ac-Cha-Gpg-Tic-Nle- 440 90 290 N(H)CH2CH2OCH2CH2OHP77 4 tetrahydroisoquinoline Ac-Cha-Gpg-Tic-Nle- 8,700 1,900 3,600tetrahydroisoquinoline P53 7 NMe2 Ac-Cha-Gpg-Tic-Nle- 410 340 180βPhPro-[SΨ(oxaz)L]-NMe₂ P82 4 N(H)CH2CH2Ph Ac-Cha-Argr-Tic-Nle- 18,0003,100 14,000 N(H)CH₂CH₂Ph P98 4 N(Bn)CH2CH2OCH2CH2OH Ac-Cha-Arg-Tic-Nle-4,700 640 1,300 N(Bn)CH₂CH₂OCH₂CH₂OH P71 4 N(Me)BnAc-Cha-Arg-Tic-Nle-N(Me)Bn 11,000 880 6,600 P81 4 N(CH2CH2OH)CH2CH2PhAc-Cha-Arg-Tic-Nle- 22,000 3,300 20,000 N(CH2CH2OH)CH2CH2Ph P88 4N(CH2CH2Pn)CH2CH2OCH2CH2OH Ac-Cha-Arg-Tic-Nle- 10,000 2,000 5,700N(CH2CH2Ph)CH2CH2OCH2CH2OH P83 4 N(Me)CH2CH2Pn Ac-Cha-Arg-Tic-Nle-24,000 1,300 11,000 N(Me)CH2CH2Ph P51 7 NMe2 Ac-Cha-Arg-Tic-Nle- 670 400210 βPhPro-[SΨ(oxaz)L]-NMe₂ P72-1 4 N(Me)Bn Ac-Cha-Arg-Disc-Nle-N(Me)Bn5,200 1,200 1,700 P78-1 4 N(Me)Bn Ac-Cha-Gpg-Disc-Nle-N(Me)Bn 2,300 5501,400 P81-1 7 NMe2 Ac-Cha-Gpg-Disc-Nle- 260 220 90βPhPro-[S(oxaz)L]-NMe2 Immunsupresssive assays IC50 (uM) and Max %inhibition) Cell binding IC50 (uM) DR4-LN DR4-LN DR14-LN DR14-LN Thyb1Thyb1 Number Priess (0401) LG2 (0101) (IC50) (Max) (IC50) (Max) (IC50)(Max) P67 4.5 4.5 NT NT NT NT 115 33 P102 NT NT 110 60 73 100 81 76 P602.1 1.5 29 97 41 98 30 47 P1 11 8.8 64 41 NT NT >200 NM P99 NT NT 110 50105 100 97 62 P85 NT NT NT NT NT NT 100 74 P96 NT NT NT NT NT NT NT NTP84 NT NT NT NT NT NT 97 100 P33 32 4.6 40 100 32 101 25 99 P69 11 6.219 101 23 107 42 96 P101 NT NT 69 100 46 100 57 88 P74 9.2 6.1 16 102 1497 47 103 P78 NT NT 36 97 36 100 118 50 P68 12 7.2 18 102 17 105 39 106P70 8.6 5.7 42 54 NT NT 83 41 P77 NT NT 10 100 14 99 73 42 P53 3 3.5 40100 32 101 25 99 P82 NT NT 27 100 NT NT 78 100 P98 NT NT 69 67 >200 NM79 66 P71 11 6.9 19 102 15 98 78 101 P81 NT NT NT NT NT NT 39 96 P88 NTNT 105 100 37 100 14 100 P83 NT NT NT NT 13 100 46 100 P51 NT 3 38 10052 99 62 88 P72-1 7.5 12 31 103 29 95 88 105 P78-1 9.8 4.6 34 103 NT NT64 106 P81-1 3.4 5 63 99 43 112 32 89 Stability (Arbitrary units) Rat,Mouse or Human plasma; Cathepsins Rat Rat Human Human Mouse Mouse Cath BCath D Number 6 h 24 h 6 h 24 h 6 h 24 h 4 h 4 h P67 10 34 97 100 71 1658 113 P102 131 121 110 116 105 101 116 120 P60 134 124 68 120 111 95 88108 P1 11 0 94 65 NT 5 87 102 P99 80 62 104 96 71 13 106 107 P85 111 71NT 129 98 57 108 104 P96 83 45 117 64 108 82 91 106 P84 104 99 131 131111 53 69 101 P33 114 108 110 122 117 185 97 99 P69 41 42 105 93 115 8592 100 P101 73 81 106 107 107 94 100 106 P74 91 71 114 125 148 97 105104 P78 184 107 73 130 90 113 100 108 P68 7 0 85 39 49 13 52 105 P70 8365 116 90 105 82 110 112 P77 129 57 111 114 141 181 107 95 P53 114 108110 122 117 115 97 99 P82 65 16 94 72 79 31 105 108 P98 71 60 116 121 8754 104 104 P71 89 53 107 90 117 67 97 104 P81 88 76 104 109 107 70 99 99P88 77 55 133 123 125 128 94 101 P83 69 85 NT NT 93 58 100 98 P51 81 60119 129 116 136 103 103 P72-1 80 67 99 99 97 75 98 101 P78-1 80 84 93107 93 18 100 104 P81-1 151 112 151 162 87 84 105 106

TABLE 4 In vivo inhibition by co-immunisation of T cell activation ofcertain compounds of the invention and certain of their Arg-containingequivilents % Reduction of response to Compound Pos. 2 HEL PPD(HEL) OVAPPD(OVA) P51 Arg 43.1  3.5 P53 Gpg 68.4 10.2 56.3 (59)   26.8 (34.4) P82Arg 25   5  P69 Gpg 59 (88)  2 (32) 65.3 (51.7)  9 (15) P71 Arg 78.162.1 48.8 21.8 P74 Gpg 51.6 (52.6) 12.8 (3.3)  42.5  5.8 P41-1 Gpg 50.121.6 P57 Gpg 52.6 52.4 P60 Gpg 50.3 28.1 P61-1 Gpg 37.9 32.3  3.7 29.9P68 Gpg −3.2 25.1 95.3 65.9 P70 Gpg 56.1 52.3 P101 Gpg 49.7 (31.7) 41.1(21.8) P102 Gpg 41.3 31.2

1. A compound having a structure of Formula II:

wherein, as valence and stability permit, A is absent or represents asequence of from one to four amino acid or amino acid analog residues; Brepresents a sequence of from two to eight amino acid or amino acidanalog residues; W represents OR₇ or NR₈R₉, V, independently for eachoccurrence, represents C═O, C═S, or SO₂; X is absent or represents O, S,or NR; R, independently for each occurrence, represents H or loweralkyl; R₁ represents a substituted or unsubstituted alkyl, heteroalkyl,alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl,cycloalkylalkyl, heterocyclyl, or heterocyclylalkyl moiety; R₂represents a substituted or unsubstituted alkyl, heteroalkyl, alkenyl,alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl,cycloalkylalkyl, heterocyclyl, or heterocyclylalkyl moiety, preferably ahydrophobic moiety, or R₂ and R, taken together, form a ring having from5 to 7 members, preferably from 6 to seven members, optionally beingsubstituted with from 1 to 5 substitutents and/or forming a polycyclicstructure with one or more other rings, such as aryl, heterocyclyl, orcarbocyclyl rings; i represents an integer from 0-1; j represents aninteger from 1-2; k represents an integer from 1-3; and R₆ is absent orrepresents from 1-4 substitutents on the nitrogen-containing ring towhich it is attached, selected from substituted or unsubstituted loweralkyl, haloalkyl, halogen, hydroxyl, and amino. R₇, R₈ and R₉independently represent substituents selected from H and substituted orunsubstituted alkyl, heteroalkyl, aryl, aralkyl, heteroaralkyl,heteroaryl, cycloalkyl, cycloalkylalkyl, heterocyclyl, andheterocyclylalkyl, or where R₈ and R₉, taken togther, form a ring havngfrom 5 to 7 members, optionally being substituted with from 1 to 5substitutents and/or forming a polycyclic structure with one or moreother rings, such as aryl, heterocyclyl, or carbocyclyl rings
 2. Acompound of claim 1, wherein R₆ is absent.
 3. A compound of claim 1,wherein at least one of R₁, R₇, R₈, and R₉ is a hydrophobic residue. 4.A compound of claim 1, wherein A represents 0 or 1 amino acid or aminoacid analog residues.
 5. A compound of claim 1, wherein B representsfrom 2 to 6 amino acid or amino acid analog residues.
 6. A compound ofclaim 1, wherein B represents from 2 to 4 amino acid or amino acidanalog residues.
 7. A compound of claim 1, wherein the compound has animmunosuppressant activity.
 8. A compound of claim 1, wherein thecompound inhibits MHC-mediated activation of T cells.
 9. A compound ofclaim 1, wherein the amino acid residues are alpha-amino acid residues.10. A compound of claim 1, wherein R₁XV, taken together, represent anacyl group.
 11. A compound of claim 10, wherein the a cyl group is analkyl carbonyl group, an aryl carbonyl group, or an aminoalkyl carbonylgroup.
 12. A compound of claim 10, wherein the acyl group is a benzoylgroup, a lower alkanoyl group, or a lower aminoalkanoyl group.
 13. Acompound of claim 10, wherein the acyl group is an acetyl group.
 14. Acompound of claim 1, having a structure selected from the structuresgiven in Table 1a or constructed from the Gpg contained preferredsubunits of Table 1b.
 15. The compound of claim 1, wherein said compoundis selected from P60, P53, P47, P41-1, P69, P66-1, P76-1, P67, P74, P80,P111 and P102
 16. The compound of claim 1, wherein said compound bindsto MHC class II protein.
 17. The compound of claim 16, wherein said MHCclass II protein is a DR molecule.
 18. The compound of claim 16, whereinsaid MHC class II protein is selected from 0401, 0101 and
 0404. 19. Thecompound of claim 16, wherein said compound binds to said MHC class IIprotein with an IC₅₀ better than one selected from 4 μM, 2 μM, 1 μM, 500nM, and 200 nM.
 20. The compound of claim 1, wherein said compound showsstability in mouse serum after 24 hours of greater than at least one of50%, 60%, 70%, 80%, and 90%.
 21. The compound of claim 1, wherein saidcompound shows stability in rat serum after 24 hours of greater than atleast one of 10%, 20%, 40%, 50%, 60%, 70%, 80%, and 90%.
 22. Apharmaceutical composition comprising a compound of claim 1 and apharmaceutically acceptable excipient.
 23. A use of the compound ofclaim 1 in the preparation of a pharmaceutical composition.
 24. The useof claim 23, wherein the pharmaceutical composition is for treating orpreventing a condition characterized by MHC class II-mediated activationof T cells or expression of an MHC class II protein at a pathologicalsite of inflammation.
 25. The use of claim 24, wherein the condition isselected from rheumatoid arthritis, juvenile arthritis, multiplesclerosis, Grave's disease, insulin-dependent diabetes, narcolepsy,psoriasis, systemic lupus erythematosus, ankylosing spondylitis,allograft rejection, transplant rejection, graft vs. host disease,Hashimoto's disease, myasthenia gravis, pemphigus vulgaris, thyroiditis,glomerulonephritis, pancreatitis, primary biliary cirrhosis, Sjogrensyndrome, scleroderma, polymyositis, dermatomyositis, bullouspemphigoid, Goodpasture's syndrome, autoimmune hemolytic anemia,pernicious anemia, idiopathic thrombocytopenic purpura, insulitis,irritable bowel disease, and Addison's disease.
 26. The use of claim 24wherein the condition is selected from rheumatoid arthritis and multiplesclerosis.
 27. A method for suppressing an immune response, comprisingadministering to an animal the compound of claim
 1. 28. A compoundhaving a structure of Formula I:

wherein, as valence and stability permit, A is absent or represents asequence of from one to four amino acid or amino acid analog residues; Brepresents a sequence of from two to eight amino acid or amino acidanalog residues; X is absent or represents O, S, or NR; W represents OR₇or NR₈R₉, V, independently for each occurrence, represents C═O, C═S, orSO₂; R, independently for each occurrence, represents H or lower alkyl;R₁ represents a substituted or unsubstituted alkyl, heteroalkyl,alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl,cycloalkylalkyl, heterocyclyl, or heterocyclylalkyl moiety; R₂represents a substituted or unsubstituted alkyl, heteroalkyl, alkenyl,alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl, cycloalkyl,cycloalkylalkyl, heterocyclyl, or heterocyclylalkyl moiety, or R₂ and R,taken together, form a ring having from 5 to 7 members, optionally beingsubstituted with from 1 to 5 substitutents and/or forming a polycyclicstructure with one or more other rings; R₃ represents a substituted orunsubstituted, alkyl, heteroalkyl, alkenyl, alkynyl, aryl, aralkyl,heteroaryl, heteroaralkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, orheterocyclylalkyl moiety; and R₇, R₈ and R₉ independently representsubstituents selected from H and substituted or unsubstituted alkyl,heteroalkyl, aryl, aralkyl, heteroaralkyl, heteroaryl, cycloalkyl,cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl, or where R₉ andR₉, taken togther, form a ring havng from 5 to 7 members, optionallybeing substituted with from 1 to 5 substitutents and/or forming apolycyclic structure with one or more other rings, wherein W includes atleast 6 non-hydrogen atoms.
 29. A compound of claim 30, wherein at leastone of R₈ and R₉ is selcted from benzyl, phenethyl, 2-hydroxyethyl, and—CH₂CH₂OCH₂CH₂OH.